Figure 10.

Adaptor-protein 2 regulates the hTfR1 cleavage activity of PC7. A, Western blot analysis of cell lysates (cells) and media (med) after overexpression of hTfR1-V5 with hPC7 and AP-1μ or AP-2μ in HEK293 cells. Normalization was performed using hTfR1-V5 cleavage by hPC7 as a reference. These results are representative of three independent experiments. B, Western blot analysis of cell lysates (cells) and media (med) after overexpression of hTfR1-V5 and hPC7 with siRNA control (scramble) or with siRNA against AP-1μ or AP-2μ in HEK293 cells. siRNA silencing was quantified by quantitative PCR. These results are representative of three independent experiments. C, co-immunoprecipitation assay of HEK293 cells transfected with a plasmid expressing Flag-tagged AP-2μ and hPC7-V5 or empty vector as a negative control. Immunoprecipitation (IP) was performed with beads coupled to an anti-Flag antibody. Cell lysates (input) and immunoprecipitates were analyzed by Western blotting (WB) with an anti-V5 antibody or anti-Flag antibody. These results are representative of three independent experiments. Error bars indicate averaged values ± S.D. *, p < 0.05; ***, p < 0.001; ns, not significant (Student's t test).