Western blot analyses were performed as described previously (Elliott et al., 2018 ; Vo et al., 2013 (link); Zhong et al., 2012 (link)). Briefly, protein samples (30–35 µg proteins) were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, Massachusetts). After blocking, the membranes were incubated with appropriate dilutions of specific primary antibodies (1:1000 for p-AKT, AKT, p-EGFR, EGFR,; 1:500 for Giα2; 1:5000 for β-actin: 1:3000 for α-tubulin) overnight at 4°C. After washing, the blots were incubated with appropriate secondary antibodies and developed in ECL mixture. The blots were then exposed to an X-ray film and visualized by autoradiography. The density of specific protein bands was determined by ImageJ software (NIH, Bethesda, MD) and normalized using density of β-actin or α-tubulin bands which were used as loading controls. For RGS2, protein samples (40 µg) were loaded on 15% SDS-PAGE gel, electrophoresed, and transferred to an Immobilon-FL membrane (Millipore). Primary antibodies were used to identify RGS2, HA tag and loading control β-actin. IRdye700- or IRdye800-labeled secondary antibodies were used for protein band detection. The images were captured with a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) at wavelengths of 700 or 800 nm.