Anti actin

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 7.4, 5 mM EDTA and protease inhibitor cocktail solution (Roche)), and lysates were cleared by centrifugation. For each sample, 40 mg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Membranes were blocked in 5% skim milk powder in TBST (0.1% Tween-20) for 1 h and incubated with the following primary antibodies overnight at 4 °C: anti-iNOS (1:500, Santa Cruz); anti-p-ERK, anti-ERK, anti-p-AKT, anti-AKT, anti-p-AMPKα/β, and anti-AMPKα/β (1:1000, Cell Signaling); anti-HIF-1α (1:200, Santa Cruz); anti-Nrf2 (1:200, Santa Cruz); anti-HSP60, anti-HSP70, and anti-HSP90 (1:500, Santa Cruz); anti-LC3 (1:1000, MBL) or anti-actin (1:2000, Sungene Biotech). After washing in TBST, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (Zhongshanjinqiao Corp.) at a 1:2000 dilution and then washed three times with TBST. Each membrane was placed into ECL solution (Thermo), and signals were subsequently detected using a Molecular Imager ChemiDoc XRS⁺(BioRad) and analyzed using Image Lab 4.0.1.

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.