C57BL/6 male mice (6-8 weeks of age) and Ifnar1 −/− mice were from the Jackson Laboratory. All mice experiments were performed in accordance with guidelines from the University of California, Los Angeles, Institutional Animal Care and Use Committee. HEK293T and RAW264.7 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. RXRαfl/fl (WT) and LysM-Cre +/RXRαfl/fl (Rxra−/−) mice bone marrows were overnight shipped from Dr Mercedes Ricote's Lab (Spain)13 (link). WT, Ifnar1 −/− and Rxra −/− BMMs were differentiated as described previously45 (link). WT and Rxra −/− F9 embryocarcinoma cells, as well as RXR-specific agonists AGN194204 and LG268 were obtained from Dr Peter Tontonoz's Lab (University of California, Los Angeles). HX531 was obtained from Dr Hiroyuki Kagechika's lab (Tokyo Medical and Dental University). 9cRA and 13cRA were purchased from Sigma-Aldrich. All compounds for in vitro treatment were solubilized in DMSO. PolyI:C and polydA:dT were from InvivoGen. Antibody against α-tubulin was from Sigma-Aldrich. Anti-VSV-G (P5D4) and anti-Oct1 (C-21) antibodies were from Santa Cruz Biotechnology. Anti-RXRα (D6G10), anti-β-Actin (13E5) and anti-β-Catenin (D10A8) were from Cell Signaling Technology. Anti-GAPDH (GT239) was from GeneTex.
Antibody against α tubulin
Manufactured by Merck Group
Sourced in United States
The Antibody against α-tubulin is a laboratory reagent used to detect and study the alpha subunit of the tubulin protein, a key structural component of the cytoskeleton in eukaryotic cells. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to visualize and analyze the distribution and expression of α-tubulin within cells.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit hrp conjugated secondary antibodies, by Cytiva (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Gfp trap coupled agarose beads, by Proteintech (2 mentions)
Poly 1 c, by InvivoGen (1 mentions)
Anti gapdh gt239, by GeneTex (1 mentions)
Ifnar1 mice, by Jackson ImmunoResearch (1 mentions)
C57bl 6 male mice, by Jackson ImmunoResearch (1 mentions)
Anti β actin 13e5, by Cell Signaling Technology (1 mentions)
Anti β catenin d10a8, by Cell Signaling Technology (1 mentions)
Poly da dt, by InvivoGen (1 mentions)
Antibody against erα, by Santa Cruz Biotechnology (1 mentions)
Anti mir 26a, by GenePharma (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Protease inhibitor cocktail, by BD (1 mentions)
Fas cd95, by Proteintech (1 mentions)
Bcl xl, by Santa Cruz Biotechnology (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Peyfp c1, by Takara Bio (1 mentions)
Pecfp c1, by Takara Bio (1 mentions)
Penicillin streptomycin, by Biowest (1 mentions)
17 protocols using antibody against α tubulin
1
Murine cell differentiation protocol
2
Detecting Xenopus Ink4d Protein
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Embryos were lysed in ice-cold lysis buffer (120 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% NP-40, 1 mM EDTA, and Complete protease inhibitors [Roche]). Lysates were cleared of lipid and yolk by Freon extraction (http://spot.colorado.edu/~klym/ ) and protein concentration was determined using a BCA Protein Assay Kit (Pierce). Equal amount of protein was resolved on 15% (w/v) polyacrylamide-SDS gels and transferred onto nitrocellulose membranes. To detect Xl-Ink4d protein, we raised a rabbit polyclonal antibody to the C-terminal peptide of Xl-Ink4d1 (amino acid sequence: SQLAAILDPRLASIFELST) and affinity-purified the antibody using the same peptide. This peptide is unique to the predicted Xenopus Xl-Ink4d protein and its sequence is shared between Xl-Ink4d1, Xl-Ink4d2 and the Ink4d of X-Tropical is but not those of Fugu, Mouse or Human. Thus the antibody does not cross react with mouse p19Ink4d protein (negative data not shown). Membranes were probed with an antibody against α-tubulin (Sigma) as a control for protein loading. Goat anti-rabbit HRP-conjugated secondary antibodies (Amersham) and Super Signal Dura (Pierce) were used to develop the blots.
3
Detecting Xenopus Ink4d Protein
4
Antibody-based Protein and miRNA Analysis
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Antibody against ERα was from Santa Cruz (Santa Cruz, CA, USA), antibody against α-tubulin from Sigma (St Louis, MO, USA). All other reagents were from Sigma. Anti-miR-26a and nonsense of anti-miR were from GenePharma (Shanghai, PR China). Cel-miR-39 mimics were from IBS (Shanghai, PR China).
5
Western Blot Analysis of Apoptosis Markers
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Cells were harvested and lysed in cell lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM sodium orthovanadate, 1X protease inhibitor cocktail (BD Bioscience, San Jose, USA)). Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane (Amersham biosciences, Waltham, Massachusetts, USA). The membrane was blocked with 5% skim milk (BioShop, Burlington, Canada) for 1 h at room temperature (RT), then probed with the appropriate primary antibodies. Antibodies against BID, BAX, Bcl-2, Bcl-XL, and FADD were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Caspase-3, Caspase-8, PARP, horseradish peroxidase (HRP) conjugated anti-rabbit, anti-mouse, and anti-rat antibodies were purchased from Cell Signaling Technology (Beverly, USA). Antibody against α-tubulin was purchased from Sigma-Aldrich, and Fas/CD95 was purchased from Proteintech, and Flag was purchased from Stratagene. The secondary antibodies were diluted at a 1:2000 ratio in 5% skim milk and incubated for 3 h at RT. Chemiluminescence signals were detected by a chemiluminescent system (Intron).
6
Expression and Characterization of TPC6A and WWOX Constructs
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Human TPC6A and TPC6AΔ were constructed in pECFP-C1, pEGFP-C1 and pEYFP-C1 (Clontech, Mountain View, CA, USA), respectively, as described.1 (link) Clones included the full-length TPC6A, TPC6AΔ and mutant TPC6AΔ(S35G) and TPC6AΔ(Y112F). Expression constructs of full-length and truncated WWOX, including WW domain, SDR domain, D3 tail and SDR/D3 region, were made as previously described.7 (link),15 (link),38 ,39 (link) Transient gene expression of indicated cDNA expression constructs by electroporation was performed as described.7 (link),15 (link),38 ,39 (link) Specific antibodies against WWOX, pY33-WWOX, TIAF1, TPC6A, TPC6AΔ and phosphorylation at pSer35 were made as described.7 (link),8 (link),15 (link),20 (link),34 ,38 ,39 (link) Antibody against α-tubulin was from Sigma (St. Louis, MO, USA).
7
EGFR Signaling and Lipid Dynamics
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HTO was obtained from Medalchemy (Alicante, Spain). The RPMI medium, triolein, RNaseA and propidium iodide were purchased from Sigma-Aldrich (St. Louis, USA), with penicillin-streptomycin purchased from Biowest (Nuaillé, France) and the fetal bovine serum (FBS) from Biosera (Boussens, France). The recombinant human EGF was obtained from R&D systems (Minneapolis, USA), while sodium orthovanadate was from Sigma-Aldrich (St. Louis, USA) and the protease inhibitors were from Roche (Roche, Basel, Switzerland). NBD-sphingomyelin and NBD-glucosylceramide were purchased from Larodan (Solna, Sweden), and NBD-ceramide from Invitrogen, Molecular Probes. The antibodies used to probe Western blots were raised against LC3BI-II, ERK, phospho-ERK (p-ERK), Akt and phospho-Akt (p-Akt S473), and they were purchased from Cell Signaling (Danvers, MA, USA). The antibody against α tubulin was purchased from Sigma-Aldrich (St. Louis, USA) and the antibodies against EGFR and p-EGFR (Y1068) were from Abcam (Cambridge, UK). The anti-EGFR antibody 930 used to stain the cell surface and to assess internalization was kindly provided by Genentech (San Francisco, USA).
8
Immunoprecipitation of GFP-fusion Proteins
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Yeast extracts prepared by alkaline extraction as described previously (Wen et al. 2010 (link)) were resolved on SDS-PAGE before subjected to Western blotting using anti-GFP antibody (Abcam) to reveal GFP-fusion proteins. Antibody against HA was from Roche. Anti-Flag M2 antibody was from Sigma-Aldrich. Antibody against α-tubulin (Sigma-Aldrich) was used as control. Anti-ubiquitin antibody was from Millipore.
For immunoprecipitation, 2 × 108 cells were lysed in 200 μL NP-40 buffer (6 mM Na2HPO4, 4 mM NaH2PO4, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, Complete protease inhibitor cocktail) by vortexing with acid-washed glass beads. The lysate was clarified by centrifugation and GFP-fusion proteins were retrieved using GFP-Trap coupled agarose beads (Chromotek), following the manufacturer's instructions.
For immunoprecipitation, 2 × 108 cells were lysed in 200 μL NP-40 buffer (6 mM Na2HPO4, 4 mM NaH2PO4, 1% NP-40, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF, 1 mM DTT, Complete protease inhibitor cocktail) by vortexing with acid-washed glass beads. The lysate was clarified by centrifugation and GFP-fusion proteins were retrieved using GFP-Trap coupled agarose beads (Chromotek), following the manufacturer's instructions.
9
Visualizing Microtubule Organization
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KMS34 cells were treated with 5 µM TC11 for 4 h and attached to a slide using CYTOSPIN4 (Thermo Fisher Scientific, Rockford, IL), then fixed with 2% paraformaldehyde and permeabilized with 0.5% Triton X followed by blocking with 1% bovine serum albumin (BSA) in PBS. The sample was stained with antibody against α-tubulin (Sigma-Aldrich) followed by FITC-conjugated anti-mouse IgG (Takara Bio, Shiga, Japan), and then observed by fluorescence microscopy (BZ-9000).
10
Nuclear Protein Extraction and Western Blot
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Briefly, frozen samples were homogenized in ice-cold buffer as described previously8 (link)16 (link). Nuclear fractions were suspended in buffer containing 20 mM HEPES, 840 mM NaCl, 0.5 mM MgCl2, 4 mM EDTA, 10% glycerol, protease and phosphatase inhibitor cocktail tablets (Roche). Protein concentrations from nuclear fractions were determined by utilizing the BCA assay kit (Thermo Fisher Scientific, Rockford, IL). Samples were then electrophoretically transferred on to PVDF membranes and incubated overnight at 4 °C with a specific antibody against HDAC2 (1:1000) (Cell Signaling). Membranes were then re-probed with an antibody against α-Tubulin (1:6000) (Sigma, St. Louis, MO). Protein signal intensity was measured on the with the Kodak image station 4000 pro (Kodak, Rochester, NY) using the Carestream Molecular Imaging software.
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