P akt pser473

Cells samples were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH7.5) with protease inhibitors (Roche, Basel, Switzerland) and 1 mM Na₃VO₄. Protein concentration of cell lysate was estimated by BCA (Thermo Fisher, Bedford, MA, USA). Cell lysates were separated on SDS-PAGE Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes (Life Technologies). The membrane was blocked in 5% non-fat milk in TBST (Tris buffered saline supplemented with Tween-20)for 1 h at room temperature and then incubated with primary antibody rabbit anti-NRBP2 (1:1000, Proteintech, Manchester, UK), p-AKT pSer473 (1:000, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (1:1000, Cell Signaling, MA, USA), mouse monoclonal BAK1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal Bax (1:500, Santa Cruz Biotechnology), β-Actin (1:500, Santa Cruz Biotechnology), mouse monoclonal Ki67 (1:400, Santa Cruz Biotechnology) overnight at 4 °C. The membrane was washed in TBST and further incubated with peroxidase-conjugated secondary antibody (anti-mouse 1:10,000, anti-rabbit 1:1000, GE Healthcare). The peroxidase activity was detected using either the Amersham ECL Western blotting detection kit (GE Healthcare) or the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermofisher Scientific, Waltham, MA, USA).

Afatinib was kindly provided by Boehringer‐Ingelheim (Ingelheim am Rhein, Germany). Gefitinib, cetuximab, and bevacizumab were purchased from EVELETH (Eveleth, MN, USA). Osimertinib was purchased from Selleck Chemicals (Houston, TX, USA). Rabbit antisera against EGFR, phospho‐specific (p) EGFR (pY1068), mitogen‐activated protein kinase (MAPK), pMAPK (pT202/pY204), Akt, pAkt (pSer473), cleaved caspase‐3, BIM, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐Ki‐67 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐CD31 antibody was purchased from DIANOVA (Hamburg, Germany).