Fig. 6. Depletion of Alix does not inhibit EGF receptor downregulation. HeLa cells treated with control RNA (–) or with siRNA (+) against Alix (see Fig. 1B) or with siRNA (+) against HCRP1. The cells were stimulated for 30 minutes with EGF 200 ng/ml (time 0), and then washed and chased for 6 hours in the presence of 10 µg/ml cycloheximide before the cells were lysed in lysis buffer. The lysates were analyzed by SDS/PAGE and sequential blotting with antibodies against EGF receptor, Alix and HCRP1. The same membrane was then reblotted with anti-tubulin to verify equal loadings. The experiment was repeated three times with the same result.