Immunoblot Analysis of CCL5-Mediated Signaling

Immunoblot assay was performed as previously described.^{51 (link)} GBM cells were treated with recombinant human CCL5 (10 ng/mL, R&D) together with or without the AKT inhibitor MK-2206 (Selleckchem, 500 nmol/L), with or without the DNA-PKcs inhibitor KU-57788 (Selleckchem, 1 μmol/L). To block the function of CCR5, MVC was added 1 h before pericyte CM stimulation. Antibodies used for immunoblot assays included anti-AKT (Cell Signaling Technology, #4685, 1:1000), anti-p-AKT-Ser473 (Cell Signaling Technology, #4060, 1:1000), anti-γ-H2AX (Cell Signaling Technology, #9718, 1:1000), anti-DNA-PKcs (Cell Signaling Technology, #38168, 1:1000), anti-p-DNA-PKcs-Ser2056 (Cell Signaling Technology, #68716, 1:1000), anti-GAPDH (Proteintech, #60004, 1:10,000).