Cells were washed once with ice cold PBS on ice, then lysed in 2x Laemmli Sample buffer, scraped and sonicated. All of the samples were boiled for 6 minutes at 100°C and analyzed by Western blotting. Protein samples were separated by 10% SDS-PAGE and transferred to 0.20 µM pore size nitrocellulose membrane with Trans-Blot® Turbo™ Transfer System, 1.3 A, 25V for 10 minutes. The membranes were then blocked with 5% (w/v) nonfat dry-milk powder solution in TBS-Tween 20 (TBST) and incubated with specific primary antibodies overnight at 4°C. The primary antibodies used in this study were as follows: anti-Hsp90 (Cell Signaling Technology, Cat# 4874), anti-caveolin-1 (Cell Signaling Technology Cat# 3238), β-tubulin (BD Biosciences, Cat# 556321), anti-dynamin II (BD Biosciences, Cat# 610263). After incubation with the primary antibodies, the membranes were washed three times for 10 minutes with TBST and incubated with horseradish-peroxidase (HRP) conjugated anti-mouse (Cell Signaling Technology, Cat# 7076) or anti-rabbit (Cell Signaling Technology, Cat# 7074) secondary antibodies. Immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) using autoradiography films. ImageJ software (Research Services Branch, National Institute of Health (Bethesda, MD) was used for densitometric analyses.
β tubulin
Manufactured by BD
Sourced in United States
β-tubulin is a protein that is a component of the cytoskeleton in eukaryotic cells. It is a key structural element of microtubules, which are involved in various cellular processes such as cell division, intracellular transport, and the maintenance of cell shape and motility.
Automatically generated - may contain errors
Lab products found in correlation
Oligomycin, by Merck Group (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (2 mentions)
Rotenone, by Merck Group (2 mentions)
Methyl pyruvate, by Merck Group (2 mentions)
Anti caveolin 1, by Cell Signaling Technology (1 mentions)
Anti hsp90, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ripa buffer, by Elpis Biotech (1 mentions)
Cocktail 2, by Merck Group (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Anti mouse igg horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Insp3r3, by BD (1 mentions)
Latrunculin b lat b, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
6 protocols using β tubulin
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Signaling Molecules
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Cells were washed in PBS and lysed in RIPA buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein phosphorylation states were preserved through the addition of phosphatase inhibitors (Cocktail II, Sigma-Aldrich) to NP-40 buffer. Protein concentrations were determined using a BCA assay kit (Pierce, Rockford, IL, USA). The proteins (10 μg/sample) were resolved through SDS-PAGE and subsequently transferred to a nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% skim milk prior to Western blot analysis. Chemiluminescence was detected using an ECL kit (Advansta Corp., Menlo Park, CA, USA) and the Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). Primary antibodies against the following proteins were used: phospho-STAT3 (Tyr705), STAT3, β-actin, MMP2, MMP9, PARP, p105/p50, p100/p52, p65, and Rel-B (Cell Signaling Technology, Beverly, MA, USA); phospho-JAK2 (Tyr221) and JAK2 (Bioss, Woburn, MA, USA); CD55 (Biorbyt, Woburn, MA, USA); β-tubulin (BD Biosciences, San Diego, CA, USA); and E-cadherin, N-cadherin, Snail, and CD97 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
3
Western Blot Analysis of Sirt1, Opa1, and β-tubulin
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The sample protein (30 µg) was separated using 7.5-15% SDS-PAGE and transferred onto a nitrocellulose membrane. Blots were blocked in 5% non-fat dry milk in TBS/Tween-20 for 1 h at room temperature before incubation with the appropriate primary antibodies against Sirt1 (1:1,000; cat. no. 9475; Cell Signaling Technology, Inc.), Opa1 (1:1,000; cat. no. 612606; BD Biosciences) or β-tubulin (1:1,000; cat. no. HC101-01; TransGen Biotech) overnight at 4°C. The blots were then incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:2,500; cat. no. 7074; Cell Signaling Technology, Inc.) or horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:2,500; cat. no. 7076; Cell Signaling Technology, Inc.) for 1 h at room temperature. Finally, protein bands on the membrane were visualized using enhanced chemiluminescent western blot detection reagent (EMD Millipore). Densitometry analysis was performed using ImageJ version 1.46 (National Institutes of Health).
4
Analyzing Cellular Signaling Pathways
5
Subcellular Signaling Pathway Analysis
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From Cell Signaling Technology: Phospho-α1-AMPK (Thr172), α1-AMPK, IP3R-1, p53, acetyl-p53, cortactin, Drp1. From BD Laboratories: β-tubulin, IP3R-3. From Sigma: MCU, acetyl-cortactin. From Abcam: Tomm20. Secondary antibodies conjugated with peroxidase were purchased from GE Healthcare Life Science. TMRE, Fluo-4, mitotracker, BAPTA-AM, BCECF-AM, DAPI, and secondary antibodies conjugated with Alexa 488, 546, 633 were from Molecular Probes. Chemicals: methyl-pyruvate, FCCP, oligomycin, rotenone, nicotinamide (NMN), latrunculin B (LatB), cytochalasin D (CytD), and Hanks’ balanced salt solution were purchased from Sigma. EX527 and compound C were purchased from Tocris Bioscience. Xestospongin B was extracted and purified from the marine sponge Xestospongia exigua as described before (Jaimovich et al., 2005 (link)).
6
Antibody Utilization for Cellular Analysis
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