a HEK293T cells co-transfected with Myc-SHKBP1 and Flag-CIN85 plasmids were lysed 48 hours after transfection. Immuno-precipitations were performed with anti-Myc antibody and control mouse IgG (mIgG). Immuno-precipitated proteins were processed for western blot with anti-Flag antibody. The membrane was stripped and re-blotted with anti-Myc antibody.
b HEK293T cells co-transfected with Flag-CIN85-3SH3 or Flag-CIN85-PCC and Myc-SHKBP1 plasmids were lysed 48 hours after transfection and cell lysates were immuno-precipitated with anti-Flag antibody and control mIgG. Immuno-precipitated proteins were processed for western-blot with anti-Myc antibody. The membrane was stripped and re-blotted with anti-Flag antibody.
c HEK293T cells transfected with Myc-SHKBP1 or its mutants and Flag-CIN85 plasmids were starved in serum-free DMEM for twelve hours and then treated with 100 ng/ml hEGF for 10 minutes. The cells were lysed and immuno-precipitated with anti-Myc antibody. Immunoprecipitated proteins were processed for western-blot with anti-Flag antibody and the membrane was then stripped and re-blotted with anti-Myc antibody.