Figure 7.

BIG2+BIG1 and AP-1 knockdown cells show a similar phenotype in terms of retrograde transport of CD4-furin. (A) HeLa cells mock-treated or treated with a pool of siRNAs for the μ1A subunit of the AP-1 complex were subjected to immunoblot analysis for detection of the γ-adaptin subunit of the AP-1 complex and the μ3A subunit of the AP-3 complex as a control. (B) HeLa cells stably expressing CD4-furin were mock-treated (a and a′) or treated with a pool of siRNAs for μ1A (b and b′) or BIG1+BIG2 (c and c′). Subsequently, the cells were incubated with anti-CD4 antibody alone (a–c) or a combination of the antibody and AlexaFluor488-conjugated EGF (a′–c′) at 19°C for 60 min, then chased for 60 min at 37°C and processed for detection of the CD4 antibody. (C) Colocalization of the internalized CD4-furin and EGF in B (bottom panels), was quantitatively estimated as described under the legend for Figure 4C. **p < 0.01.