HER3 Activation and Inhibition Kinetics

2 × 10⁶ cells/dish were cultured at 37 °C for 24 h. After serum starvation in RPMI/1%serum with antibiotics for 24 h, cells were incubated with various compounds. For short kinetics, cells were stimulated with 100 ng/ml of NRG1 and/or incubated with 50 μg/ml of 9F7-F11 for 15 min to 3 h. For long kinetics, cells were incubated with 50 μg/ml of anti-HER3 antibody 9F7-F11 for 24 h to 120 h. After incubation, cells were washed, scraped and lysed with CHAPS buffer (Sigma-Aldrich), as indicated above. After washing in 1X PBS, the insoluble fraction was removed by centrifugation, and protein concentration in cell lysates was determined using the BCA assay. Two hundred micrograms of protein lysates were directly mixed with Laemmli buffer and heated at 95 °C for 5 min. After electrophoresis in reducing conditions, proteins were transferred to polyvinylidenedifluoride membranes (Millipore) and then incubated with the relevant primary and peroxidase-conjugated secondary antibodies, as previously described [33 (link)]. β-tubulin or β-actin were used as loading control.