Figure 4

Hrs is not required for recruitment of Eps15 to GFP-FYVE-UbΔGG or endogenous ubiquitinated cargo. COS-7 cells were transfected with siRNA targeting Hrs or control siRNA, and also with plasmids encoding FLAG-Eps15 and GFP-FYVE-UbΔGG. A. Proteins in lysates (equal volumes from each sample) were separated by SDS-PAGE and then analyzed by Western blotting, probing with anti-Hrs antibodies (top) or anti-Hsp70 antibodies as a loading control (bottom). B. Bands on the blot shown in Panel A were quantitated by densitometry, normalizing to the signal in the control sample. C. Cells transfected with the indicated constructs were processed for IF microscopy, staining with anti-FLAG and AF-594 goat anti-rabbit IgG to detect FLAG-Eps15 and with anti-Hrs and AF-647 goat anti-mouse IgG to detect Hrs (pseudo-colored blue). D, E. COS-7 cells transfected with control siRNA (D) or Hrs siRNA-2 (E) and with FLAG-Eps15 were processed for IF microscopy, detecting FLAG-Eps15 with anti-FLAG antibodies and AF-488 goat anti-rabbit IgG, and EEA1 with anti-EEA1 antibodies and AF-594 goat anti-mouse IgG. F, G. COS-7 cells co-transfected with FLAG-Eps15 together with either GFP or GFP-Rab5Q79L were processed for IF microscopy, detecting FLAG-Eps15 with anti-FLAG antibodies and AF-594 goat anti-rabbit IgG, and EEA1 with anti-EEA1 antibodies and AF-647 goat anti-mouse IgG (pseudo-colored blue). Merged images are shown at the right. Scale bars; 10 μm.