Calnexin c5c9

Cells were washed with ice-cold PBS, harvested by scraping and then homogenized in CelLytic M lysis buffer (Sigma, Merck) supplemented with phosphatase and protease inhibitors (PhosSTOP and cOmplete, Roche, Basel, Switzerland). Lysates were incubated (1 h, 4°C) and then centrifuged at 14,000 x g (10 min, 4°C). Protein samples were quantified using the Pierce BCA Protein Assay (ThermoFisher Scientific), resolved by SDS-PAGE and transferred to PVDF membranes (Millipore, Merck). Membranes were blocked with Tris-buffered saline/0.5% Tween 20 (TBST, pH 7.5) containing 5% skim milk powder and probed overnight with primary antibodies against the following targets: PSMA (1H8H5, ThermoFisher Scientific), EGFR (D38B1), HER2 (44E7), HER3 (D22C5), p-AKT(S473) (587F11), p-AKT (T308) (244F9), AKT (D38B1), p-S6 (S235/236) (D57.2.2E), Calnexin (C5C9) and Tubulin (9F3) were from Cell Signaling Technology, MA, USA, AR (Santa Cruz Biotechnology, TX, USA), GAPDH (GT239, GeneTex, CA, USA) and mGluR1 (07-617, Merck). Following incubation with horseradish peroxidase-linked secondary antibodies (Dako, Agilent Technologies, CA, USA), immunoreactive bands were visualized with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific).