Figure 4.

Block in retrograde transport of CD4-furin at late endosomes by depletion of BIG2 and BIG1. HeLa cells stably expressing CD4-furin were mock-treated or treated with a pool of siRNAs for BIG1, BIG2, or BIG1+BIG2 as indicated. Subsequently, the cells were incubated with anti-CD4 antibody (Leu3a) alone (A and D) or a combination of the antibody and AlexaFluor488-conjugated EGF (B) at 19°C for 60 min to allow the antibody and fluorescent EGF to accumulate in early endosomes (top panels), then chased for 60 min at 37°C (bottom panels). The cells were incubated with anti-EEA1 (A) or anti-LBPA (D) antibody and then with secondary antibodies. In C, colocalization between the internalized CD4-furin and the EGF in the bottom panels in B was estimated as described in Materials and Methods. *p < 0.05, **p < 0.01. In D, the Golgi signal of CD4-furin markedly decreased due to optimizing the immunofluorescence staining for LBPA.