For protein expression analyses, iWAT was homogenized in TNET buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) as described (Morley et al., 2015 (link)) with Halt protease and phosphatase inhibitors (Thermo Pierce). Samples from tissue lysates were then resolved by SDS-PAGE. Immunoblotting was performed using standard protocols. Membranes were blotted with the following antibodies: anti-FASN (BD Biosciences); anti-UCP1 (Abcam); anti-TH (Millipore); anti-phospho HSL-S660 and anti-phospho perilipin (Cell Signaling Technology), anti-Actin and anti-Tubulin (Sigma-Aldrich).
Anti actin and anti tubulin
Manufactured by Merck Group
Anti-Actin and anti-Tubulin are laboratory reagents used for the detection and localization of actin and tubulin proteins in cells. Actin and tubulin are key structural components of the cytoskeleton, which plays a crucial role in cell shape, motility, and division. These reagents are commonly used in immunocytochemistry, western blotting, and other cell biology techniques to investigate the distribution and dynamics of the cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Anti th, by Merck Group (2 mentions)
Anti ucp1, by Abcam (2 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Anti phospho hsl s660 and anti phospho perilipin, by Cell Signaling Technology (2 mentions)
Anti fasn, by BD (2 mentions)
Geneticin g418 sulfate, by Thermo Fisher Scientific (1 mentions)
Anti phospho chk1 ser345, by Cell Signaling Technology (1 mentions)
Anti rad51, by Merck Group (1 mentions)
Cycloheximide (chx), by MP Biomedicals (1 mentions)
Chk1 inhibitor ucn01, by Merck Group (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Anti traf6 d 10, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti actin and anti tubulin
1
Protein Expression Analysis in iWAT
2
Protein Expression Analysis in iWAT
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For protein expression analyses, iWAT was homogenized in TNET buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) as described (Morley et al., 2015 (link)) with Halt protease and phosphatase inhibitors (Thermo Pierce). Samples from tissue lysates were then resolved by SDS-PAGE. Immunoblotting was performed using standard protocols. Membranes were blotted with the following antibodies: anti-FASN (BD Biosciences); anti-UCP1 (Abcam); anti-TH (Millipore); anti-phospho HSL-S660 and anti-phospho perilipin (Cell Signaling Technology), anti-Actin and anti-Tubulin (Sigma-Aldrich).
3
Western Blot and Immunofluorescence Assays
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The following chemicals were used in the study: ATR inhibitor (VE-821) and ATM inhibitor (KU-55933) from Selleckchem, CHK1 inhibitor (UCN-01) from Sigma, Geneticin (G418 sulfate) from Thermo Fisher Scientific, and Cycloheximide (CHX) from MP Bio. The following antibodies were used in western blot analysis and immunofluorescent staining: anti-53BP1 and anti-FANCJ from Novus Biologicals, anti-phospho-CHK1 (Ser345) from Cell Signaling, anti-RAD51 from Millipore, anti-CHK1, anti-E2F6 and anti-E2F1 from Santa Cruz Biotechnology, anti-Actin and anti-Tubulin from Sigma, ECL HRP-linked secondary antibody from GE healthcare. Rabbit polyclonal anti-TOPBP1 and anti-BRCA1 antibodies were home-raised as previously described (56 (link),57 (link)). Anti-RRM2 serum was raised in rabbit against an immunogen containing the first 250 amino acids of human RRM2. Dr. Kai Ge provided the antibody against PTIP.
4
Antibody Generation and Protein Interactions
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HGK antibody was generated by immunization of rabbits with individual peptides (HGK epitope: HAPEPKPHYDPADRAREV). Anti-actin and anti-tubulin antibodies were purchased from Sigma. Anti-p-IRS-1 (S612; 2386), anti-p-IRS-1 (Y896; 3070) and anti-IRS-1 (#2382) antibodies were purchased from Cell Signaling. Anti-TRAF1 (H-3), anti-TRAF2 (H-10), anti-TRAF3 (M-51), anti-TRAF4 (C-20), anti-TRAF5 (C-19) and anti-TRAF6 (D-10) antibodies were purchased from Santa Cruz. All first antibodies for immunoblotting were used in 1:1,000 dilution. The expression plasmids for HGK and HGK kinase-dead mutant were as described previously8 (link). CFP-tagged HGK, Flag-tagged TRAF2 and YFP-tagged TRAF2 were constructed by subcloning the individual complementary DNA into pCMV6-AC-CFP, pCMV6-AC-Flag and pCMV6-AC-YFP vectors (OriGene Technologies), respectively. The TRAF2 short hairpin RNA plasmid was established by the National RNAi Core Facility (Taiwan) and its repeat insertion (5′-GCGATCTTCATCAAAGCTATT-3′) was subcloned into pSUPER-GFP vector (OligoEngine, Inc.). Lysotracker was purchased from Molecular Probes.
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