Anti tubulin

The shRNA targeting human GLUL mRNA (5′-GACAATGCCCGACGTCTAA-3′) was cloned into pLKO.1-GFP lentiviral vector. Full-length of GLUL-WT/or R324C, N-Cadherin and deletion mutants were cloned into pDest27-GST or pCDH-Neo-Venus/Dest vectors as previously described²⁸ (link). Anti-GLUL (Cat#D122427) and anti-GFP (Cat#D110008) antibodies were purchased from Sangon Biotech (Shanghai, China). Anti-N-Cadherin (Cat#610920), anti-β-Catenin (Cat#610154), anti-c-Myc (Cat#551102) and anti-Cyclin D1 (Cat#514181) antibodies were purchased from BD Bioscience (NJ, USA). Anti-Vimentin (Cat#5741) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GST (Cat#SC-459) and anti-HA (Cat#SC-7392) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Anti-Flag (Cat#F7452) antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-GAPDH (Cat#KM9002T), anti-β-actin (Cat#KM9001T) and anti-Tubulin (Cat#KM9007T) antibodies were purchased from Sungene Biotech (Tianjin, China).

Cells were lysed in RIPA buffer (Genestar) supplemented with protease inhibitor cocktail (MCE) and proteins were extracted after centrifugation of the supernatant at 12,000×g and 4 °C. Proteins were loaded into an electrophoresis chamber for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred to nitrocellulose membranes. Membranes were blocked in TBST containing 5% skim milk powder for 1 h and were then incubated with the following primary antibodies overnight at 4 °C, anti-HSPA8 (Abcam, Cat. # ab51052); anti-P16 (ABclonal, Cat. # A0262), anti-P21 (ABclonal, Cat. # A19094), anti-P38 (Cell Signaling Technology, Cat. # 9212S), anti-pp38 (Cell Signaling Technology, Cat. # 9211S), LaminB1 (Sino Biological, Cat. # 101237-T32), anti-actin (Sungene Biotech, Cat. # KM9001) and anti-tubulin (Sungene Biotech, Cat. # KM9003T). After washing with TBST, membranes were incubated for 1 h at room temperature with the corresponding secondary antibody. After washing with TBST three times, membranes were incubated with ECL solution (Thermo) and chemical signals were acquired using a ChemiDoc XRS + molecular imager (BioRad). The band signals were analyzed with Image Lab software.