Chapter 16 - Investigating Tubulin Posttranslational Modifications with Specific Antibodies

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Abstract

Microtubules play highly diverse and essential roles in every eukaryotic cell. While built from conserved dimers of α- and β-tubulin, microtubules can be diversified by posttranslational modifications in order to fulfill specific functions in cells. The tubulin posttranslational modifications: acetylation, detyrosination, polyglutamylation, and polyglycylation play important roles in microtubule functions; however, only little functional and mechanistic insight has been gained so far. The modification state of microtubules can be visualized with specific antibodies. A drawback is that detailed information about the specificities and limitations of these antibodies are not easily accessible in the literature. We provide here a comprehensive description of the currently available set of antibodies specific to tubulin modifications. Focusing on glutamylation antibodies, we discuss specific protocols that allow using these antibodies to gain semi-quantitative information on the levels and distribution of tubulin modifications in immunocytochemistry and immunoblot.

Introduction

Microtubules (MTs) are core components of a complex filamentous system called the cytoskeleton. Present in every eukaryotic cell, they display a variety of functions including the regulation of cell shape, polarity and motility, cell division, as well as intracellular transport. Moreover, MTs are essential regulators of cell differentiation, as for instance in neurons. They are also the major building blocks of cilia, flagella, and centrosomes, where they assemble into highly complex structures called axonemes and centrioles. Considering the diversity of MT structures and associated functions, it is obvious that mechanisms are needed to create different MT identities.

In the past, great advances have been made in understanding the implication of MT-associated proteins (MAPs) and molecular motors in the functional specialization of MT populations. Various MAPs decorate discrete populations of MTs in a single cell, and motors selectively bind to MT subspecies (e.g., certain MT populations of the mitotic spindle, or axonal vs. dendritic MTs in neurons). However, in many of these well-studied systems, the signals that confer specificity to these specific MAP–MT interactions are not completely understood.

Two mechanisms have been considered as sources of MT diversity: the incorporation of different tubulin isoforms, also known as isotypes, and the posttranslational modifications (PTMs) of MTs. Of these two mechanisms, PTMs are the regulators that are specifically added to already assembled MTs and can be easily controlled in a spatial and temporal manner. It is not so for tubulin isoforms that can only slowly, by changing the gene expression profiles, get incorporated into newly formed MTs. Thus, tubulin PTMs, which include acetylation, detyrosination, polyglutamylation, and polyglycylation, are the prime candidates for fine-tuning MT functions (Box 16.1; reviews: Janke and Bulinski, 2011, Luduena, 2013).

Despite the variety of functions tubulin PTMs are expected to fulfill, current research in the MT field often fails to correctly assess the importance or even the presence of these modifications. Functional studies have been difficult because most of the enzymes catalyzing tubulin PTMs had not been discovered until recently (Akella et al., 2010, Janke et al., 2005, Kimura et al., 2010, Rogowski et al., 2009, Rogowski et al., 2010, Shida et al., 2010, van Dijk et al., 2007, Wloga et al., 2009). Moreover, tubulin PTMs are hard to quantify or even detect. For instance, glutamylated peptides are often not detected in classical mass spectrometry, a problem that has been overcome for purified tubulin, but that requires relatively large amounts of protein (Redeker, 2010). Furthermore, modifications do not significantly alter the migration properties of tubulin on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); hence, gel-shift cannot be used to assay these modifications.

Thus, the most reliable way for distinguishing differentially modified MTs and tubulin in tissues, cells, and by immunoblot is PTM-specific antibodies. While some of these antibodies have proven to be excellent tools, precise data about their specificities and limitations have not been comprehensively reported. Here, we present a collection of well-analyzed antibodies specific to tubulin PTMs, together with appropriate protocols that allow using them in a semi-quantitative fashion in immunofluorescence on cells, as well as in immunoblot. We illustrate the opportunities and limitations of the described methods based on the example of glutamylation-specific antibodies.

We have found that the cell fixation method is crucial for the correct preservation of the MT cytoskeleton, as well as for the quality of detection with some of the modification-specific antibodies. Neither a paraformaldehyde (PFA)–sucrose fixation nor the fixation with cold methanol provides satisfying results, especially for the staining of MT modifications of certain structures, as for instance the midbody. Therefore, we use a method in which the cytoskeleton is prefixed with a bifunctional protein cross-linker, dithiobis succinimidyl propionate (DSP; Bell & Safiejko-Mroczka, 1995). To demonstrate the impact of these three methods on immunolabeling of cultured cells, we have fixed HeLa cells that were cultured on fibronectin-coated coverslips and stained the cells with polyE and 12G10 antibodies. Cells in anaphase, representing a midbody structure, were observed (Fig. 16.1).

Using the DSP fixation method, we have previously shown that midbody MTs carry long glutamate side chains (Lacroix et al., 2010); however, this specific labeling is only seen when cells are fixed with the DSP–PFA method. In contrast, fixing the same cells with either the PFA or the cold methanol method prevents the detection of this polyglutamylated MT population (Fig. 16.1), indicating that polyE is sensitive to the fixation method. In contrast, in all three fixation methods, the tubulin antibody 12G10 shows a nice MT labeling (Fig. 16.1), which can be particularly misleading as it suggests that MTs have been fully preserved. A possible explanation for the divergence between polyE and 12G10 labeling on midbody MTs is the assumed low level of polyglutamylation on those MTs. Partial disassembly of MTs during PFA and methanol fixations would affect rare epitopes much more than the abundant (and anyway too dense—see Section 16.1.2) epitopes of tubulin. Though this effect has mostly been observed with polyE antibody, it is likely that other modification-specific antibodies are also sensitive to the cell fixation. We therefore strongly recommend testing different fixation conditions if specific PTMs of MTs are tested, even if the MTs are apparently intact if detected with a PTM-independent antibody, or with an antibody for a tubulin PTM that is highly enriched on the specific MTs investigated.

A MT is a hollow tube of 25 nm of external diameter and is in most organisms composed of 13 protofilaments. Each protofilament is a linear assembly of α-/β-tubulin dimers, and protofilaments assemble side by side to form the MTs. Considering that the diameter of an α- or β-tubulin molecule is about 4–5 nm, the dense assembly of MTs generates a situation where similar epitopes are arranged in a distance of about 4 nm. An average antibody has an approximate diameter of 15 nm length and width, a size that is even further increased by the binding of secondary antibodies that can take the space of a cube, the side length of which could be around 30 nm or more. Thus, it is obvious that the spatial constraints on the MT will not allow for a stoichiometric decoration of epitopes on each tubulin molecule. In other words, only a fraction of the tubulin molecules present in the MT lattice can be labeled with antibodies due to space constrains. This creates a severe problem when antibodies directed against PTMs of tubulin are used to estimate the modification status and level of a given MT population using immunolabeling in fixed cells.

While it is easy to distinguish MTs carrying very low levels of modification from those with very high levels in cells, it is much harder to see subtle differences, which could still be highly relevant from a functional point of view. Moreover, even in situations where only a given percentage of the tubulin molecules in the MT lattice are posttranslationally modified, decoration with modification-specific antibodies could generate strong and virtually continuous signals that cannot be distinguished from those obtained from MTs with much higher modification levels. To avoid misinterpretations of the modification status of MTs based on antibody labeling, it is essential to test serial dilutions for each of the antibodies used. Moreover, it is important to perform such tests for each novel experimental setting.

To illustrate the importance of dilution in the correct assessment of changes in tubulin modifications, we have transfected the U2OS cell line with either the glutamylase TTLL4, known to generate glutamylation on MTs (van Dijk et al., 2007), or the deglutamylase CCP5, known to remove glutamate side chains (Rogowski et al., 2010). Strikingly, at dilutions commonly used for the antibody GT335 (0.5–0.1 μg/ml), cytoplasmic MTs are not clearly labeled in nontransfected cells (Fig. 16.2A, white contours), while all MTs are fully labeled in TTLL4-expressing cells (Fig. 16.2A, green contours). In contrast, no change in modification levels is seen in cells transfected with CCP5 with 0.5 μg/ml GT335 (Fig. 16.2B, lower panel, green contours). However, using a higher concentration of the antibody (5 μg/ml) reveals a clear MT staining in nontransfected U2OS cells (Fig. 16.2B, upper panel, white contours), while this staining is strongly decreased in cells expressing the deglutamylase CCP5 (Fig. 16.2B, upper panel, green contours).

This demonstrates that U2OS cells carry low levels of glutamylation on their interphase MTs (Regnard, Desbruyeres, Denoulet, & Eddé, 1999), which can only be revealed at higher concentrations of GT335 antibody. This antibody concentration is, of course, too elevated for the detection of MTs with higher levels of PTMs, where even much lower concentrations of the same antibody are sufficient for strong labeling (Fig. 16.2A, lower panel). While this conclusion might appear trivial in this overexpression experiments, it is important to point out that similar situations might be encountered in cells. For instance, single MTs in cells might acquire specific functions by slight changes in MT PTMs, which could be easily overseen using antibodies at high dilutions. Conversely, in cells containing MTs with very high modification levels, such as neurons, developmental changes in MT PTM patterns can only be observed if antibodies are sufficiently diluted. To illustrate this notion, we show here the staining of a 3-day-old rat cortical neuron, stained with 0.1 mg/ml of GT335. Despite the fact that MTs in young neurons are not fully glutamylated (Audebert et al., 1994; Fig. 16.2C), and despite the high dilution of the antibody, the MTs in these neurons appear already strongly labeled, suggesting that even higher dilutions of GT335 might be required to determine changes in MT glutamylation during neuronal development.

Finally, it should be noted that not only the density of the tubulin molecules within an MT but also the density of MTs within certain cellular structures, such as neuronal axons and dendrites (Fig. 16.2C), axonemes in cilia and flagella (Bressac et al., 1995), or other MT bundles in cells, such as midbodies in anaphase cells (Fig. 16.1), preclude the possibility of identifying specific PTM identities of single MTs within these MT arrays. Because there is as yet no easy approach to circumvent this problem (even super resolution light microscopy is not able to fully resolve single MTs in dense bundles), it should be taken into account when interpreting immunostaining results from fixed cells.

Strikingly, tubulin concentration is also a critical factor in immunoblot analyses, especially if semi-quantitative information about the levels of PTMs should be deduced from the detection levels with the different antibodies. Apart from the above-discussed importance of using a range of dilutions of the antibodies to determine the optimal (or better minimal) concentration (Fig. 16.3A), another important factor is the dilution of the protein samples that are loaded on the SDS-PAGE. Because tubulin, even at relatively high concentrations, resolves into one (standard SDS-PAGE) or two (specific SDS-PAGE to resolve α- and β-tubulin; Lacroix & Janke, 2011 and described below) neat bands, overloading of the antigen is often not noticed. In contrast, differences in tubulin modification levels are easily overlooked if too much material has been loaded on the SDS-PAGE prior to immunoblot (Fig. 16.3B). As the presence of tubulin PTMs does not significantly alter the apparent molecular weight of tubulin separated on an SDS-PAGE, there is no additional hint, apart from the antibody detection levels, indicating the PTM state of tubulin.

An additional factor that can easily obscure the need to dilute the antibodies of tubulin PTMs is that most of these antibodies yield only very low background signals, even if used in high concentrations. This has been observed for both, immunocytochemistry and immunoblot (Fig. 16.3A), and could thus hamper the identification of differences in modification levels in these two experimental setups.

So far, only one site of tubulin acetylation has been studied in detail. Acetylation of lysine-40 of α-tubulin is specifically detected with the mouse monoclonal antibody 6-B11-1 (IgG2b isotype), which has been raised against the axonemal α-tubulin from sea urchin sperm flagella (Sigma #T6793, #T7451; LeDizet and Piperno, 1987, LeDizet and Piperno, 1991, Piperno and Fuller, 1985). The antibody detects acetylated tubulin in most species tested so far.

Tyrosinated α-tubulin is recognized by the YL1/2 rat monoclonal antibody (Millipore #MAB1964; Cumming et al., 1984, Kilmartin et al., 1982). This antibody was raised against purified yeast tubulin and thus recognizes the C-terminal -EEF and -EEY sequences on α-tubulin.

Anti-detyr-tubulin rabbit polyclonal antibody (Millipore #AB3201; Paturle-Lafanechere et al., 1994), raised against the -CGEEEGEE(-COOH) peptide, recognizes the C-terminal -GEE sequence of detyrosinated α-tubulin. Another antibody, the mouse monoclonal 1D5 (Synaptic Systems #302011; Rüdiger et al., 1999, Warn et al., 1990), also detects C-terminal stretches of two and more glutamate residues (− En; n  2). However, while 1D5 detects detyrosinated α-tubulin (which ends with C-terminal -EE in most species), it cross-reacts with any polyglutamylated form of tubulin that contains glutamate side chains that contain two or more glutamate residues. Thus, the use of this antibody alone makes the interpretation of the data impossible.

▵2-tubulin (see Box 16.1) can be detected by a rabbit polyclonal antibody (Millipore #AB3203; Paturle-Lafanechere et al., 1994) that has been raised against the peptide -CEGEEEGE(-COOH). Anti-▵2-tubulin antibody detects specifically C-terminal -GE sequence of proteins (Rogowski et al., 2010).

Two antibodies allow assessing the extent of both α- and β-tubulin polyglutamylation: the mouse monoclonal GT335 (Adipogen #AG-20B-0020; Wolff et al., 1992), which recognizes the epitope formed at the branching point of the glutamate side chains (γ-linked glutamate on a main-chain glutamate), and thus stains both short and long side chains of tubulin polyglutamylation. It has been raised against the branched peptide -EGEGE*EEG(-CONH2) with a bi-glutamate side chain (-EE(-COOH)) attached to the γ-carboxy group of the glutamate marked with the asterisk. GT335 also detects a range of nontubulin substrates of glutamylation (Regnard et al., 2000, van Dijk et al., 2008).

Polyglutamylation can be detected using the rabbit polyclonal antibody polyE (Rogowski et al., 2010, Shang et al., 2002, van Dijk et al., 2007). This antibody is still not commercially available. It detects stretches of at least three glutamate residues situated at the C-terminal extremity of proteins and has been raised against a -CEEEEEEEEE(-COOH) peptide, and is therefore specific to long side chains added by polyglutamylation to tubulin and other substrates, and it also detects proteins that have a genetically encoded C-terminal tail with three or more glutamate residues (Rogowski et al., 2010).

The use of these two antibodies, GT335 and polyE, allows distinguishing between long (GT335- and polyE-positive) and short (GT335-positive, polyE-negative) side chains generated by polyglutamylation.

The mouse monoclonal antibody TAP952 detects monoglycylated tubulin (Callen et al., 1994), but unlike GT335 (detecting both short and long glutamate chains), it does not cross-react with elongated glycine side chains. TAP952 has been raised against Paramecium axonemal tubulin and been shown to detect γ-linked glycine residues on glutamic acids 437, 438, 439, and 441 of peptides corresponding to C-terminal tails of paramecium β-tubulin (Bré, Redeker, Vinh, Rossier, & Levilliers, 1998). Though the antibody has been shown to detect strongly glycylated tubulin in cilia and flagella of many species, it is not yet clear whether all possible modification sites are detected, especially because of the high-sequence variability of tubulin within the C-terminal tails, where the modification takes place.

AXO49 is a mouse monoclonal antibody raised against Paramecium axonemes that recognize γ-linked glycine side chains of at least three residues (Callen et al., 1994). It has been mapped to detect side chains of three and more glycine residues attached to the Glu 437 of the paramecium β-tubulin C-terminal peptide (Bré et al., 1998).

The rabbit polyclonal polyG antibody has been raised against the -CGGGGGGGGG(-COOH) peptide and detects, similar to the polyE antibody, C-terminal polyglycine chains (Rogowski et al., 2009, Wloga et al., 2009).

None of the antibodies against glycylation are commercially available.

HeLa cells (ATCC® #CCL-2™) were grown on glass coverslips (Marienfeld #01 115 20) in DMEM (Life technologies #41965-039) supplemented with 10% fetal bovine serum (Sigma #F7524) and 2 mM l-glutamine (Life technologies #25030-024) at 37 °C and in 5% CO2. Before use, coverslips were incubated in 60-μl drops of a 5 μg/ml aqueous fibronectin (Sigma #F1141) solution for 1 h at 37 °C in a humid chamber. Coverslips were then transferred to a 24-well plate and washed gently three times with phosphate-buffered saline (PBS) and left in PBS until the seeding of cells.

The U2OS cell line (ATCC® #40342™) was grown as described above. Cells were transfected with expression vectors encoding the murine TTLL4 (van Dijk et al., 2007) or CCP5 (Rogowski et al., 2010) genes using jetPei® (Polyplus #101-10) according to manufacturers’ instructions. Cells were fixed 20 h posttransfection.

Rat cortical neurons were cultured as previously described (Saudou, Finkbeiner, Devys, & Greenberg, 1998) and let to grow until 3 days in vitro in neurobasal-B27 medium.

Coverslips were washed once with PBS, then rapidly immersed in − 20 °C methanol, and incubated at − 20 °C for 10 min. Methanol was removed, and the coverslips were washed three times with PBS and stored in PBS at 4 °C if required.

Coverslips were rinsed once with PBS and incubated in 3.7% PFA, 2% sucrose solution for 15 min at room temperature (RT). Coverslips were rinsed three times with PBS and could be stored in PBS at 4 °C if required.

This method has been described previously (Bell & Safiejko-Mroczka, 1995) and involves several incubation steps that are all performed at RT:

  • 10 min incubation in 1 mM DSP in Hank’s balanced salt solution (HBSS)

  • 10 min incubation in 1 mM DSP in microtubule stabilizing buffer (MTSB)

  • 5 min incubation in 0.5% Triton X in MTSB

  • 15 min incubation in 4% PFA in MTSB

  • 5 min wash in PBS

  • 5 min incubation in 100 mM glycine in PBS (in order to quench remaining aldehyde groups)

  • 5 min wash in PBS at RT, followed by an optional storage at 4 °C in PBS.

All the solutions should be at RT in order to preserve intact MTs inside the cells (in all steps prior to the PFA fixation, MTs depolymerize if cold solutions are used).

Coverslips with fixed cells were subjected to indirect immunochemistry. Antibodies have been diluted to the desired concentration in PBS, 3% bovine serum albumin, 0.1% Triton X-100 (this solution can be filtered, aliquoted, and stored at − 20 °C). In order to use a minimum amount of antibodies, we incubate coverslips with attached cells facing downward on a drop of 30 μl of antibody solution in a humid chamber for 1 h at RT. 12G10 is a monoclonal antibody detecting α-tubulin (Developmental Studies Hybridoma Bank, University of Iowa). C105 is a rabbit polyclonal, anti-β-tubulin antibody (gift of Jose M. Andreu, Madrid; Arevalo, Nieto, Andreu, & Andreu, 1990). The coverslips were then transferred to a 24-well plate and rinsed three times with PBS, 0.1% Triton X-100, and subsequently incubated with the fluorescent secondary antibodies as previously described (Life technologies #A11001 Alexa 488 anti-mouse IgG, #A11019 Alexa 568 anti-mouse IgG, #A11036 Alexa 568 anti-rabbit IgG, all used at 2 μg/ml, and #A21068 for Alexa 350 anti-rabbit IgG used at 10 μg/ml). The coverslips were again transferred to a 24-well plate and rinsed four times with PBS, 0.1% Triton X-100 (if DNA staining was required, the first wash was performed in PBS, 0.1% Triton X-100, 0.1 μg/ml DAPI for 3 min at RT). Coverslips were then mounted carefully on microscopy slides using 6 μl of Mowiol solution. Mowiol is left to polymerize by leaving the slides in horizontal position at RT and in the dark for at least 4 h, and most commonly overnight.

Images were acquired on a Leica DMRXA upright microscope using Metamorph (Molecular Devices) software. Images were treated using ImageJ (http://imagej.nih.gov/ij/) or Photoshop (Adobe Systems).

We use a variation of SDS-PAGE allowing for the separation of α- and β-tubulins as two distinct bands. The method has been described in detail in Lacroix and Janke (2011). It differs from the standard SDS-PAGE protocol by a different acrylamide to bis-acrylamide ratio, the pH of the Tris buffer, and the type of SDS (see “buffer composition,” Section 3.2.1).

Brains of either 2 days old or adult mice were rapidly homogenized in Laemmli sample buffer and using an Ultra-Turrax® homogenizer (IKA®) and heated to 95 °C for 5 min. Subsequently, two short pulses of sonication were applied to fragment genomic DNA.

The samples were loaded onto the SDS-PAGE gels (for tubulin, 10% acryl amide gels are used) and run until the bromophenol blue front reaches the bottom of the gel. The gels were then shortly incubated in transfer buffer, and proteins were transferred onto a nitrocellulose membrane (Protran BA 85, Whatman, GE Healthcare #10 401 196) for 1 h at 4 °C at 100 V and approximately 400 mA using a tank blot system (Bio-Rad #170-4070).

After transfer, the nitrocellulose membranes were incubated for 1 min in Ponceau S staining solution and destained twice for 2 min in 1% acidic acid. The membranes were scanned and transferred to Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). Subsequently, the membrane was incubated in 5% fat-free dry milk in TBS-T at RT for 1 h. The primary antibodies were diluted in 2.5% milk–TBS and were incubated with the membrane at RT for 1 h. After three washes of 5 min with TBS-T, the membranes were transferred into a dilution of secondary, horseradish peroxidase (HRP)-labeled antibody (anti-mouse- or anti-rabbit-HRP-conjugated IgG; GE Healthcare #NA931V or #NA934V, respectively) in TBS-T and incubated at RT for 45 min. After five washes of 3 min with TBS-T, membranes were developed by incubation within the ECL Western Blotting Detection Reagents (GE Healthcare #RPN2209) for 1 min. The membrane was then exposed to photographic films (Amersham Hyperfilm™ MP, GE Healthcare #28906844) for 1 min. The films were developed using a photographic developing machine (Curix60, Agfa) and scanned with trough light using a linear gray scale gradient.

  • PFA–sucrose in PBS

    • Prepare 4% PFA (Sigma #P6148) in PBS (first dissolve in water by adding NaOH, bring to pH 7.0, and adjust the concentration with 10 × PBS) and then use this solution to prepare 3.7% PFA, 2% sucrose solution in PBS (can be aliquoted and stored at − 20 °C).

  • DSP stock solution

    • DSP 20 mg/ml in DMSO (50 mM; Perbio Pierce #22585) can be aliquoted and stored at − 20 °C.

  • HBSS: Hank’s balanced salt solution (can be prepared as 5 × buffer and stored at 4 °C); 1 × solution can be obtained from Gibco (#14025-050)

    • 1.26 mM CaCl2

    • 5.33 mM KCl

    • 0.44 mM KH2PO4

    • 0.5 mM MgCl2

    • 0.41 mM MgSO4

    • 138 mM NaCl

    • 4 mM NaHCO3

    • 0.3 mM Na2HPO4

    • 5.6 mM glucose

  • MTSB: Microtubule stabilizing buffer (store at 4 °C)

    • 1 mM EGTA

    • 4% PEG 8000

    • 100 mM PIPES, pH 6.9

  • TSB

    • 0.5% Triton X-100 in MTSB

  • PFA in MTSB

    • 4% PFA in MTSB: PFA powder (Sigma #P6148) is heated in MTSB to approximately 50 °C and dissolved by adding a drop of 10 M NaOH; the pH is then brought to pH 7.0 with 6 M HCl. The solution is left to cool down to RT.

  • Mowiol mounting medium

    • 10% Mowiol (polyvinyl alcohol) 4-88 (Sigma #81381)

    • 100 mM Tris/HCl, pH 8.5

    • 25% glycerol

  • 5 × Laemmli sample buffer

    • 450 mM DTT

    • 10% SDS (BDH #442444H)

    • 400 mM Tris/HCl, pH 6.8

    • 50% glycerol

    • bromophenol blue

Electrophoresis cell: Mini-PROTEAN® cell with a PowerPac™ power supply (Bio-Rad #165-8001EDU and #164-5052MP, respectively).

  • Separating gel buffer (4 ×)

    • 1.5 M Tris/HCl, pH 9.0

    • 0.4% SDS (specific type required: Sigma #L5750)

  • Stacking gel buffer (4 ×)

    • 0.5 M Tris/HCl, pH 6.8

    • 0.4% SDS (Sigma #L5750)

  • Acrylamide/bis-acrylamide stock solution (74:1)

    • 40% acrylamide solution (Bio-Rad #161-0140) supplemented with 0.54% bis-acryl amide (w/v) powder (Bio-Rad #161-0210).

  • Running buffer

    • 50 mM Tris/HCl

    • 384 mM glycine

    • 0.1% SDS (Sigma #L5750) in deionized water

  • Transfer buffer

    • 50 mM Tris/HCl

    • 40 mM glycine

    • 0.4 mM SDS

    • in deionized water

    • 20% (v/v) ethanol

  • Ponceau S staining solution

    • 0.2% (w/v) Ponceau S in 1% acetic acid

Section snippets

Concluding Remarks

Here, we have summarized our experiences in the detection of tubulin PTMs using immunocytochemistry and immunoblot. We have described appropriate methods to detect changes in MT PTMs using a battery of modification-specific antibodies and suggest several experimental precautions to take in order to avoid misleading interpretations. Because many functional aspects of MT PTMs might be related to gradual changes rather than to abrupt changes, careful evaluation of changes in MT modification

Acknowledgments

This work was supported by the Institut Curie, the CNRS, the INSERM, the Labex CelTisPhyBio 11-LBX-0038, the French National Research Agency (ANR) awards 08-JCJC-0007, the Human Frontier Science (HFSP) grants RGP0023/2008, the Institut National du Cancer (INCA) grant 2009-1-PL BIO-12-IC-1, and an EMBO Young Investigator Program grant to C. J.

We thank C. Benstaali, P. Marques (Institut Curie, Orsay, France), A. Fleury-Aubusson (CBM, Gif-sur-Yvette, France) for technical assistance, and R. Basto,

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      2016, Cell
      Citation Excerpt :

      Indeed, PTMs including phosphorylation, detyrosination, polyglutamylation, polyglycylation, and acetylation are enriched on specialized microtubule structures such as centrioles and basal bodies, neuronal axons, and primary cilia (Janke, 2014; Song and Brady, 2015). Most microtubule PTMs have been discovered serendipitously, usually as the result of generation of antibodies later found to react with specific modified residues of α- or β-tubulin (Magiera and Janke, 2013), and as a result, the complete repertoire of microtubule PTMs has yet to be fully elucidated. PTMs of microtubules serve many functions, such as recognition by microtubule-associated proteins (MAPs), which can regulate microtubule dynamics and function.

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