Anti t akt antibody

Protein lysate was extracted and the protein concentration was determined by the BCA method (Biyuntian, Nantong, China). Total 30 μg protein in each condition was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose membranes (Immobilon-P, Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl and 1‰ Tween-20) at room temperature for 1 h and incubated with the anti-P-Akt (Ser473) antibody (1:1000 dilution, Cell Signaling Technology, Beverly, Mass, USA), anti-T-Akt antibody (1:1000 dilution, Cell Signaling Technology, Beverly, Mass, USA), pan anti-acetyllysine antibody (1:1000 dilution, PTM Biolabs, Hangzhou, China), anti-integrin β5 antibody (1:500 dilution, Abcam, MA, USA), anti-acetyl-Histone H2B (Lys12) antibody (1:2000 dilution, PTM Biolabs, Hangzhou, China), and anti-GAPDH antibody (1:5000 dilution, Kangchen bio-tech, Shanghai, China) at 4 °C overnight. After washing with TBST, the membranes were reacted with the peroxidase-conjugated affiniPure goat anti-rabbit or goat anti-mouse secondary antibodies (1:5000 dilution, Zhongshanjinqiao, Beijing, China) for 1 h at room temperature. After extensive washing with TBST, signals were detected using enhanced chemiluminescent reagents (Thermo Scientific, IL, USA).

For Western blot analysis, podocytes were lysed in hypotonic lysis buffer (Beyotime), and equal amounts of protein were denatured after heating at 95°C for 5 min and loaded on to an SDS/8% polyacrylamide gel. Separated proteins were subsequently transferred to a nitrocellulose membrane and blocked with 8% nonfat milk at room temperature for 1 h. Membranes with proteins were incubated with primary antibodies at 4°C overnight. The primary anti-MR antibody (1:100, Santa Cruz Biotechnology), anti-human integrin β1 polyclonal antibody (1:400, Bosider), anti-p85-PI3K antibody (1:800, Cell Signaling Technology), anti-p-Akt antibody (ser473, 1:800, Cell Signaling Technology), anti-t-Akt antibody (1:800, Cell Signaling Technology), anti-p-mTOR antibody (ser2448, 1:200, Santa Cruz Biotechnology), anti-Atg5 antibody (1:500, Santa Cruz Biotechnology) and β-actin antibody were used. The second antibodies (horseradish peroxidase conjugate goat anti-rabbit, 1:5000 in blocking solution) were added and incubated for 2 h at 4°C. All blots were developed using Western blotting detection system of enhanced chemiluminescence (Pierce Biotechnology).