Anti α tubulin

HEK293T or Hep3Bcells were cultured in DMEM or MEM (Gibco) medium containing 10% fetal bovine serum (Hyclone). All assays were performed as described before (15 (link)). The used primary antibodies were: anti-Myc and anti-HA from Santa Cruz; anti-Flag from Sigma; anti-Smad2/3, anti-phospho-Smad2(Ser-465/467), and anti-phospho-ERK1/2 (Thr-202/Tyr-204) from Cell Signaling Technology; anti-ERK1 from OriGene; anti-pAKT (Ser-473) and anti-p-Jun1/2(Thr-183/Tyr-185) from Cell Signaling Technology, anti-β-actin from Santa Cruz, and anti-α-tubulin from EasyBio.

Different cells were harvested after treatment and proteins were extracted to detect the expression by Western blotting as previously described with minor modifications [46 (link)]. Equal amounts of proteins were size fractionated by 9 to 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Anti-SIRT6 (2590 S, Cell Signaling Techology, Danvers, MA, USA), anti-KAT8 (ab200660, abcam, Cambridge, MA, USA), anti-Acetyl-lysine (PTM-105RM, PTMBIO, China), anti-Flag (F1804, Sigma Aldrich), anti-SIRT1 (8469 S, Cell Signaling Techology, Danvers, MA, USA), anti-SIRT7 (5360 S, Cell Signaling Techology, Danvers, MA, USA), H4K16ac (ab109463, Abcam Cambridge, MA, USA), anti-RNA Pol II (sc-47701, Santa Cruz, CA, USA), anti-E-cadherin (14472 S, Cell Signaling Techology Danvers, MA, USA), anti-N-cadherin (13116 S, Cell Signaling Techology Danvers, MA, USA), anti-Histone H4 (ab177840, Abcam, Danvers, MA, USA), anti-His (PM032, MBL), anti-GST (sc-138, Santa Cruz, CA, USA), anti-GCN5 (sc-365321, Santa Cruz, CA, USA), anti-myc (M047-3, MBL), anti-α-tubulin (BE0031, EASYBIO, Beijing, China) and anti-β-actin (4967, Cell Signaling, Danvers, MA, USA) were used and the blots were developed using an enhanced chemiluminescence kit (Amersham Corp.).

Cells were lysed in buffer containing 50 mM Tris pH8.0, 2 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol and protease inhibitor PMSF. Western blot analysis was carried out based on standard methods. The following commercial antibodies were used as probes: anti-Flag antibody (Sigma, F3165), anti-HA antibody (Sigma, H3663), anti-α-Tubulin (EasyBio, BE0026–100), anti-PKM2 (RuiYingBio, RLT3777), anti-TOM 40 (Santa Cruz sc-365467), anti-succinyllysine antibody (PTM BIO PTM-901), anti-GAPDH (SanJian).