Anti pegfr

The Western blot analysis was performed as previously described. U87-EGFRvIII cells were cultured in DMEM supplement with 10% FBS at 37°C in a 5% CO2/95% air atmosphere. The cells were planted in a 6-well plate and treated with DMEM, AuNP or U2-AuNP, respectively. After 24h, cells were lysed in RIPA buffer supplemented with protease inhibitors. After protein quantification with a BCA protein assay kit (Thermo Scientific), 20µg protein was detected by 8% SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were incubated with the following specific antibodies: anti-pEGFR (tyr1038), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pH2AX (S139), anti-pChk2 (T68), anti-Chk2 (Cell Signaling Technology, Danvers, MA), anti-pATM (Ser1981), anti-ATM (Cell Signaling Technology, Danvers, MA), anti-53BP1 (Cell Signaling Technology, Danvers, MA) and anti-β-actin (Boster Biological Technology, China). A secondary HRP-conjugated antibody was used for immunodetection, and an ECL Western blotting kit (Bio-Rad) was used to visualize the protein bands. Finally, the results were analyzed with ImageJ software.