Anti gapdh g8795

Anti-FLAG (F7425), anti-Myc (M4439) and anti-GAPDH (G8795) antibodies were purchased from Sigma (St. Louis, MO, USA). Anti-His₆ (AE003) was purchased from Abclonal (Shanghai, China). [³H] SAM, [³H] Arg, [¹⁴C] Thr and [¹⁴C] Ser were obtained from Perkin Elmer Inc. (Waltham, MA, USA). Dynabeads Protein G, MitoTracker and Lipofectamine 2000 transfection reagent were obtained from Thermo Scientific (Waltham, MA, USA). Primers were synthesized in Biosune (Shanghai, China), and DNA sequencing was performed by Tsingke (Shanghai, China).

Tissue lysates in buffer containing 0.05% NP-40, 50 mM NaCl, 0.5 mM EDTA, 50 mM Tris-HCl (pH 7.4), and 10 mM NAM with 1X EDTA-free protease inhibitors (Roche Life Sciences, Basel, Switzerland) were electrophoresed on Criterion SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. The primary antibodies used were anti-Sirt2 (ab67299; Abcam), anti-GAPDH (G8795; Sigma-Aldrich, St. Louis, MO, USA), anti-c-MYC (Cat # 5605; Cell Signaling, Danvers, MA, USA), anti-Alpha tubulin (Cat # 66031-1-Ig, Proteintech, Rosemont, IL, USA), and anti-Lamin A/C (Cat # 10298-1-AP; Proteintech). After incubation with 1:10,000 Goat anti-Rabbit-HRP IgG (STAR208P; Bio-Rad) or Goat anti-Mouse-HRP IgG secondary antibody (STAR207P; Bio-Rad), the blots were visualized with Clarity Max ECL (BioRad), scanned, and in some cases subjected to densitometric analysis using ImageJ software.

Western blotting was carried out as described previously [20 (link)]. In brief, PMSF-RIPA lysis buffer was added to the cells and the resultant lysate was centrifuged at 12,000 rpm for 5 mins to permit collection of the supernatant. The concentration of each protein was measured by the BCA method and the concentrations were adjusted to separation by SDS-PAGE. For each sample, we loaded 15 μl (50 μg) per well. Following separation, proteins were transferred to the nitrocellulose membrane. Membranes were then incubated with primary antibodies (anti-CD9, CBL162, Sigma-Aldrich; anti-TSG101, SAB2702167, Sigma-Aldrich; anti-GAPDH, G8795, Sigma-Aldrich; anti-PPARγ, MAB3872, Sigma-Aldrich; anti-Col2, CP18, Sigma-Aldrich; anti-Runx2, AV36678, Sigma-Aldrich; anti-Sox9, AV37986, Sigma-Aldrich; and anti-Aggrecan, MABT83, Sigma-Aldrich) at 1 : 1,000 and secondary antibodies at 1 : 5,000 (goat anti-rabbit (ab6721, Abcam) or goat anti-mouse (ab97023, Abcam) horseradish peroxidase- (HRP-) conjugated secondary antibody), and positive binding was visualized using a ChemiDoc™ XRS imaging system (Bio-Rad, Beijing, China). The immunoreactive bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).