Anti acetylated α tubulin

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-acetylated-α-tubulin is a primary antibody that binds to acetylated α-tubulin, a modification of the cytoskeletal protein α-tubulin. This antibody can be used to detect and visualize acetylated microtubules in cells.

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6 protocols using anti acetylated α tubulin

Comprehensive Protein Expression Analysis

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Western blotting has previously been described (18 (link)). Anti-pEIF2α^S51, anti-EIF2α, anti-ATF4, anti-GRP78, anti-IRE1α, anti-acetylated-α-tubulin, anti-BCL2, anti-BCLXL, anti-PUMA, anti-BID, anti-BIM (Cell Signaling Technology, Beverly, MA, USA) and anti-pIRE1α^S724 (Abcam, Cambridge) were used in conjunction with a HRP-conjugated anti-rabbit secondary antibody (Amersham, Buckinghamshire, UK). Anti-caspase-8 (12F5; Alexis, San Diego, CA, USA), anti-CHOP (Cell Signaling Technology), anti-ATF6 (Abcam), anti-MCL1 (BD pharmingen, Oxford, UK) and anti-NOXA (Abcam) mouse monoclonal antibodies were used in conjunction with a horseradish peroxidase–conjugated anti-mouse secondary antibody (Amersham).

Forsythe N., Refaat A., Javadi A., Khawaja H., Weir J.A., Emam H., Allen W.L., Burkamp F., Popovici V., Jithesh P.V., Isella C., Labonte M.J., Mills I.G., Johnston P.G, & Van Schaeybroeck S. (2018). The unfolded protein response: a novel therapeutic target for poor prognostic BRAF mutant colorectal cancer. Molecular cancer therapeutics, 17(6), 1280-1290.

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Antibody Analysis of Ciliary Proteins

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Anti-ADAMTS9 (Novus, NBP1-82916), anti-β-actin (Abcam, ab6276), anti-acetylated-α-tubulin (Cell Signaling Technology, 5335S), and anti-V5 (Cell Signaling Technology, 80076S) antibodies were used. Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Invitrogen. Horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Santa Cruz Biotechnology. At 24 h after transfection, the cells were switched to serum-free medium. After 60 h, cells were harvested and lysed. Serum-free spent medium was collected separately and concentrated using Amicon Ultra-4 (Millipore). Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes and blotted with the indicated primary antibodies. Confocal images were obtained using the Carl Zeiss LSM780 microscope. Image processing and analysis were performed using the ZEN software. For immunofluorescence, RPE1 cells were seeded at a low density, grown to confluence, and then serum-starved for 48 h to induce primary cilia. ADAMTS9 immunoperoxidase images were obtained from the Human Protein Atlas with the original source available at the following link: (https://www.proteinatlas.org/ENSG00000163638-ADAMTS9/tissue/kidney).

Yu S., Choi Y.J., Rim J.H., Kim H.Y., Bekheirnia N., Swartz S.J., Dai H., Gu S.L., Lee S., Nishinakamura R., Hildebrandt F., Bekheirnia M.R, & Gee H.Y. (2023). Disease modeling of ADAMTS9-related nephropathy using kidney organoids reveals its roles in tubular cells and podocytes. Frontiers in Medicine, 10, 1089159.

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Immunofluorescent Staining of Primary Cilia

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MEFs were plated on Nunc Lab-Tek II Glass Chamber Slides (ThermoFisher Scientific), serum-starved for 24h in serum-free media, and fixed in 4% paraformaldehyde. Cells were permeabilized in 0.2% Triton X-100 and blocked in 1% bovine serum albumin. Primary cilia were stained with anti-acetylated α-tubulin (Cat #5335, Cell Signaling Technology Inc., Danvers, MA) at 1:500 dilution followed by anti-mouse-Alexa 488 at 1:300 dilution before imaging. Nuclei were stained with 2 μg/ml DAPI solution before imaging.

Ho Wei L., Arastoo M., Georgiou I., Manning D.R, & Riobo-Del Galdo N.A. (2018). Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia. PLoS ONE, 13(8), e0203170.

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Immunostaining of MERS-CoV Infection

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Antibodies and reagents used for immunostaining are listed here: Anti-acetylated α-tubulin (Cell Signaling, cat#5335S), anti-DPP4 (R&D Systems, cat#AF1180), anti-MERS-CoV N protein (Sino Biological, cat#40068-MM10), anti-cleaved caspase-3 (Cell Signaling, cat#9664), and Alexa Fluor 647 phalloidin (ThermoFisher Scientific, cat#A22287). Methods for immunolocalization of DPP4 in human airway epithelia were adapted from previous studies (7 (link)). Briefly, airway epithelia or cytospun BAL samples were fixed in 4% paraformaldehyde and permeabilized with 0.2 % Triton X-100. Following incubation with SuperBlock™ buffer, primary antibodies were applied to the fixed cells on slides, or to the apical and basolateral cell surfaces of epithelial sheets overnight, followed by washing with SuperBlock™ buffer three times. After a 1 h incubation with the appropriate Alexa Fluor secondary antibodies, followed by more washes, coverslips were mounted using Vectashield antifade mounting medium with DAPI (Vector Laboratories). Photomicrographs were taken using a Leica TCS SPE confocal microscope. The number and types of infected cells were counted manually using ImageJ.

Li K., Wohlford-Lenane C., Bartlett J.A, & McCray PB J.r. (2022). Inter-individual Variation in Receptor Expression Influences MERS-CoV Infection and Immune Responses in Airway Epithelia. Frontiers in Public Health, 9, 756049.

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Purification and Analysis of Tachyzoites

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Infected NDHF fibroblasts were treated with SAHA and TST for 48 h, then scraped and lysed through passages in a 30G needle syringe, lysed cells were centrifuged at 2000 rcf for 10 min and resuspended in PBS and passed through an 8-μM filter to purify the tachyzoites. In total, 10⁶ parasites in a 10-μL lysis buffer containing a 0.1% protease inhibitor cocktail (Sigma) were cryolysed. Membranes were incubated with anti-α-tubulin (1:1000), anti-acetylated α-tubulin (1:2000), anti-Histone H3 (1:1000, CellSignaling), anti-acetylated Histone H3 (Lys 9/14) (1:1000, Santa Cruz), anti-Histone H4 (1:3000, CellSignaling) or anti-acetylated Histone H4 (Lys 16) (1:1000, Sigma) for 1 h depending of the assay. GAPDH was used as the loading control (1:2500, Abcam). Membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (Promega) 1:4000 in TBS-T. Membranes were then exposed to an enhanced chemiluminescence (ECL) reagent (Promega) and visualized using the ImageQuant LAS 500 (GE Healthcare Life). Quantification of band intensities from three independent experiments was performed with the NIH ImageJ software. The normalization of the amount of protein was performed using the loading control in all quantitative analyzes. The ratio of rTubac/rTub, rH3ac/rH3 and rH4ac/rH4 for each treatment was also carried out.

Araujo-Silva C.A., De Souza W., Martins-Duarte E.S, & Vommaro R.C. (2020). HDAC inhibitors Tubastatin A and SAHA affect parasite cell division and are potential anti-Toxoplasma gondii chemotherapeutics. International Journal for Parasitology: Drugs and Drug Resistance, 15, 25-35.

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Protein Extraction and Immunoprecipitation for Hypoxia Signaling

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Cells were placed on ice immediately following treatment and washed with ice-cold PBS. Wash buffer and lysis buffer contained 1x protease inhibitor cocktail (Thermo Scientific), NaF (4 mM), Na₃VO₄ (20 mM), NaCl (150 mM), β-glycerophosphate (50 mM), and DTT (0.2 mM). Nuclear and cytosolic proteins were prepared using the CellLytic NuCLEAR extraction kit (Sigma Aldrich). Immunoprecipitation was performed using Protein A/G Plus Agarose beads (Pierce) following manufacturer’s protocol using anti-HIF-1α (Abcam), anti-HDAC6 (Cell Signaling), and anti-acetyl-lysine (Cell Signaling). Total cell proteins were resolved on 8-10% SDS-polyacrylamide gels and transferred to PVDF membranes (Fisher Scientific). Membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 5% nonfat dry milk in TBST with the anti-HIF-1α (1:500, R&D Systems; 1:1000, Abcam); anti-HIF-2α (1:200, R&D Systems); anti-HDAC6 (1:1000), anti-HSP90 (1:1000), anti-acetylated-α-tubulin (1:1000) all from Cell Signaling; anti-β-tubulin (1:5000, Developmental Studies Hybridoma Bank); anti-α-tubulin (1:2000, Abcam); or anti-Lamin A/C (1:1000, Cell Signaling). Immunolabeling was detected using ECL reagent (LAS4000, GE Life Sciences). Densitometric analysis was performed using ImageQuant TL (GE Life Sciences).

Schoepflin Z.R., Shapiro I.M, & Risbud M.V. (2016). Class I and IIa HDACs mediate HIF-1α stability through PHD2-dependent mechanism while HDAC6, a class IIb member, promotes HIF-1α transcriptional activity in nucleus pulposus cells of the intervertebral disc. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 31(6), 1287-1299.

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