Peroxidase conjugated goat anti rabbit or anti mouse igg secondary antibody

Cells were washed, scraped, and resuspended in lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Proteins were solubilized by sonication, separated by SDS-PAGE, and electroblotted onto polyvinylidenedifluoride membranes (Millipore). Membranes were blocked in Tris-buffered saline and Tween-20 solution containing 5% skim milk or bovine serum albumin and then probed with a primary antibody directed against PI3Kδ (Abcam), p-AKT (Cell Signaling Technology), PD-L1 (Biolegend, San Diego, CA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing and blocking, membranes were incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at dilutions of 1:10,000 for 1 h.

Cells were washed, scraped, and resuspended in lysis buffer (iNtRON Biotechnology, Seoul, Korea). Proteins were solubilized by sonication and equal amounts of protein were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). Membranes were blocked in PBS containing 0.1% Tween 20 and 5% powdered milk and probed with primary antibody directed against p-EGFR (Tyr1068), p-Akt (Ser473), p-ERK (Tyr202/204), p-p70S6K (Thr421/Ser424), HSP70, HSP90, DNA-PKs (Thr2609), Rad51, caspase-3, LC3, MMP-2, E-cadherin, and EphA2 (Cell Signaling Technology, Inc.) at 1:1000 dilutions. Primary antibodies against MGMT (Abcam, Cambridge, UK) and Acetyl Histone H3 (Millipore Corp.) were used at a dilution of 1:1000. Antibodies against VEGF and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at dilutions of 1:500 and 1:5000, respectively. Membranes were washed and incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a dilution of 1:5000.