Anti apol1

A total of 10 6 cells for each treatment were pelleted and lysed in RIPA lysis buffer (Sigma) supplemented with protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 12000g, 4 °C for 15 min and protein concentration was measured using bicinchoninic acid assay kit (ThermoFisher). Proteins were separated by SDS-PAGE in reducing conditions and transferred to nitrocellulose membrane (GE Healthcare Life Sciences). After saturation with 5% milk, the membrane was incubated overnight at 4 °C with the appropriate primary antibody: anti-Phospho ERK1/2 1:5000, anti-total ERK 1/2 (1:10000), anti-Phospho Akt (1:5000), anti-total Akt (1:10000), anti-Phospho FAK (1:1000), and anti-total FAK (1:1000) (Abcam), anti-APOL1 (1/1000), anti-APOL2 (1/ 2000), anti-APOL3 (1/2500) and anti-APOL6 (1/500) (sigma), washed, then incubated with (HRP) conjugated secondary antibody for 1 h at room temperature and revealed using the ECL substrate (PerkinElmer). Monoclonal anti-β-actin antibody (1/80000; Sigma) was used for protein loading control. Densitometric analysis was performed using ImageJ.