Ha 11 mms 101p

Whole-cell lysate was prepared by suspending cells in the RIPA buffer (Beyotime, China) supplemented with 1× complete protease inhibitors mixture and 1× phosphatase inhibitor (Roche). Protein concentration was determined by BCA assay (Pierce, Rockford, USA). Equal quantities of proteins were separated by SDS/PAGE, transferred to a PVDF membrane, and blotted with specific antibodies. Protein in the membrane was visualized by an enhanced chemiluminescense detection kit (Millipore, USA). Rabbit antibodies against the following proteins or modifications were used with the catalogue numbers and sources indicated: CD317 (ab134061), TGF-α (ab208156) (Abcam); caspase3 (9662) (CST); pY1068 EGFR (BS5010), pY845 EGFR (BS5013), pY705 STAT3 (BS4181), STAT3 (AP0365), ERK½ (BS1112), pT202/Y204 ERK½ (BS5016), cyclin D1 (BS6352), and p16 INK4a (BS6431), caveolin-1 (BS9878M) (Bioworld Technology), and AREG (16036–1-AP) (Proteintech). Mouse antibodies against the following proteins/epitopes were used: HA.11 (MMS-101P) (Covance); Ki67 (P6834) (Sigma); transferrin receptor (13–6800) (Invitrogen); GAPDH (MB001) (Bioworld Technology); β-actin (sc-47778) and EGFR (sc-373746) (Santa Cruz Biotech). HRP-conjugated mouse anti-His (M20020) was purchase from Abmart., mouse HRP-conjugated goat anti-mouse IgG (074–1806) from KPL, and HRP-conjugated goat anti-rabbit IgG (E030120–02) from EARTHOX.

Myc-9E10 (#MMS-150P) and HA.11 (#MMS-101P) monoclonal antibodies for immunoprecipitation and immunoblotting were from Covance (now BioLegend). Flag antibodies were from either Sigma (#F3165) or Cell Signaling Technology (CST; #2368). The following antibodies were from CST: AMPK P-Thr¹⁷² (#4188), pan-AMPKα (#2532), AMPK β1 (#12063), Akt P-Ser⁴⁷³ (#4060), Akt P-Thr³⁰⁸ (#4056), Akt P-Thr⁴⁵⁰ (#9267), Akt (#9272), NDRG1 P-Thr³⁴⁶ (#3217), NDRG1 (#5196), Bad P-Ser¹³⁶ (#4366), mTOR (#2972), raptor P-Ser⁷⁹² (#2083), raptor (#2280), S6K1 P-Thr³⁸⁹ (#9234), GST (#2625), cleaved caspase 3 (#9664), cleaved PARP (#9544), Erk1/Erk2 (#9102), β-actin (#4967), and α-tubulin (#2144). mTOR P-Ser²⁴⁸¹ was from Millipore (#09–343). S6K1 P-Thr²²⁹ antibody was from Abcam (#ab59208). The following custom polyclonal anti-peptide antibodies were generated by us with the aid of Covance, as described previously (45 (link)): mTOR P-Ser¹²⁶¹ (amino acids 1256 to 1266; rat), rictor (amino acids 6 to 20; human), and S6K1 (amino acids 485 to 502; rat 70-kDa isoform). Donkey anti-rabbit–HRP secondary antibody was from Jackson ImmunoResearch (#711-095-152), and sheep anti-mouse–HRP was from GE Healthcare (#NA931V).