Fig. 5. Depletion of EHD3 or rabenosyn-5 causes impaired biosynthetic transport of cathepsin-D to lysosomes. HeLa cells, transfected with GFP-lgp120, were mock treated (A-G) or treated with EHD3 siRNA (H-K) or rabenosyn-5 (L-O) for 48 hours before fixation. TGN was detected with rabbit anti-TGN46 followed by Alexa Fluor 405-conjugated goat anti-rabbit. Anti-cathepsin-D was used to visualize endogenous cathepsin-D, followed by Alexa Fluor 568-conjugated goat anti-mouse. A-C are single-stain images of a mock-treated cell. Triple-stained images are split into pairs of colors to facilitate visualization of the distribution patterns, with the corresponding merged images (G,K,O). The graphs in P are a quantitative analysis of colocalization (see Materials and Methods) between cathepsin-D and GFP-lgp120 (left graph, representing E, I and M), whereas the right graph quantifies the colocalization of cathepsin-D with TGN46, representing F, J and N. ^*P<0.01 compared with mock-treated cells. (Q) Mock- and EHD3-siRNA-treated HeLa cells were pulsed with [³⁵S]cysteine/methionine and chased for the indicated times. The cells were lysed, and immunoprecipitated with anti-cathepsin D antibody. At the 5 hour time point, the entire supernatant was collected and immunoprecipitated with anti-cathepsin-D, to detect levels of mis-sorted cathepsin D that has been secreted from the TGN. Samples were separated by SDS-PAGE prior to exposure of the gels to autoradiograhic film. (R) Levels of cathepsin-D precursor bands were quantified by densitometry from the representative gel displayed in Q. Scale bar: 10 µm.