Fig. 9. Colocalization of endogenous Eps15 and endogenous ubiquilin to aggresomes. (A) GFP-ubiquilin and FLAG-Eps15 colocalize to aggresomes. HeLa cells transiently co-transfected with constructs encoding GFP-ubiquilin and the FLAG-Eps15 were incubated overnight with 5 µM MG132 and processed for fluorescence microscopy using the anti-FLAG antibody followed by a Cy3-labelled goat anti-mouse immunoglobulin secondary antibody. (left) Green fluorescence emitted by GFP. (right) Red fluorescence emitted by Cy3. (B) Characterization of the anti-Eps15 nanobody by western blot. Cos-1 cells were transiently co-transfected with wild-type FLAG-Eps15 and with FLAG-Eps15-I (lacking the EH 1-3 domains). Total cellular lysates were subjected to SDS-PAGE and blotted onto PVDF membrane. (left) Immunodetection with the anti-Eps15 nanobody (IB: VHH--Eps15) that was detected using the anti-Myc antibody. (right) Immunodetection with the anti-FLAG antibody (-FLAG). Notice that the llama VHH against Eps15 that was raised against domain I (EH1-3) of Eps15 does not recognize FLAG-Esp15-I as expected. (C) Characterization of the anti-Eps15 nanobody by immunoprecipitation. Biotinylated anti-Eps15 nanobody was added to HeLa cellular lysates and precipitated using streptavidin-Sepharose beads (IP: VHH--Eps15). As a control, HeLa cellular lysates were precipitated with streptavidin-Sepharose beads in the absence of antibody (Control). The beads were subjected to SDS-PAGE and immunoblotting was performed with a rabbit anti-Eps15 antibody (IB: -Eps15). Cellular lysates from HeLa were also loaded on the SDS-PAGE (Lysate). (D) HeLa cells were mocked treated (0.1% DMSO) (Control, top) or incubated overnight with 5 µM MG132 (bottom) and fixed with ice-cold methanol. Endogenous ubiquilin was detected using a rabbit anti-ubiquilin antibody revealed with Alexa-488-labelled goat anti-rabbit immunoglobulins secondary antibody (left). Endogenous Eps15 was detected using the VHH against Eps15, followed by mouse anti-Myc antibody revealed with Alexa-555-labelled goat anti-mouse immunoglobulin secondary antibody (centre). (right) Merged pictures, with yellow indicating colocalization of Eps15 and ubiquilin. The arrow indicates an aggregate in which Eps15 and ubiquilin colocalize.