Fig. 6. Kek1 association and inhibition of human EGFR. (A) Association of Kek1-HA with human EGFR in two stably transfected HEK293-Ecr cell lines. Cells were treated without or with Ponasterone A (PonA) to induce Kek1-HA expression, and lysates were immunoprecipitated (IP) with antibodies to EGFR. Precipitates were then immunoblotted with anti-HA. Cell lysate (right lane) was included as a positive control for blotting. (B,C) Inhibition of human EGFR signalling in HEK293 cells by Kek1. 293-Ecr stably transfected cells (clone 4) were treated without and with PonA for 24 hours, and then treated without and with EGF as indicated. (B) Inhibition of EGFR autophosphorylation: upper panel, lysates from treated cells were immunoprecipitated with anti-phosphotyrosine antibodies; precipitates were blotted with anti-EGFR. Lower panel, lysates were blotted with anti-HA to detect Kek1-HA expression. (C) Inhibition of Erk1/2 activation: lysates from treated cells were blotted with antibodies specific for phosphorylated Erk1 and Erk2 (upper panel) and re-probed with antibodies that recognize the total Erk2 population (middle panel). Bands were quantified and relative Erk activity plotted (lower panel).