Fig. 4. EGFR downregulation is impaired by the overexpression of SKD1(E235Q). (A) GFP-SKD1(E235Q)-expressing and control A431 cells were treated with EGF (500 ng/ml) at 37°C for the periods indicated and the lysates were subjected to immunoblotting with anti-EGFR or anti-SKD1 antibodies. The 180 kDa band indicates EGFR, while the 50 kDa and 80 kDa bands indicate endogenous SKD1 and virally expressed GFP-SKD1(E235Q), respectively. The efficiency of infection was monitored by the expression of GFP in living cells and usually exceeded 90%. (B) The remaining EGFR bands at each time point were quantitated with NIH-Image and indicated as a percentage relative to that at time 0 hours. (C) A431 cells were infected with the GFP-SKD1(E235Q)-expressing adenovirus at a lower multiplicity of infection, so as to infect about 50% of cells. Then they were treated with EGF (500 ng/ml) at 37°C for 2 hour and processed for immunofluorescence analysis with anti-EGFR antibody followed by Alexa594-labeled secondary antibody. In GFP-SKD1(E235Q)-expressing cells (asterisks), the accumulation of EGFR in E235Q compartments is shown by yellow arrowheads. Little or no EGFR is observed in uninfected cells (n.i.).