Figure 3. Rabip4’ functions in distribution of lysosomes.

The indicated cell lines were screened for expression of rabip4s by Western blot (A). HEK293T cells were transfected with siRNA against rabip4s and three days later processed for Western blot with antibodies against rabip4s and actin as a loading control, followed by Alexa680-conjugated secondary antibody. siRNA induced an >90% reduction of both rabip4’ and rabip4 isoforms (B). In parallel, cells were labeled for immunoflourescence with monoclonal antibodies against CD63, LAMP-1, CI-MPR, and TfR. LAMP-1 was counterstained with Alexa 594-, while CD63, CI-MPR, and TfR with Alexa 488-labeled secondary antibodies. Nuclei were stained with DAPI. Images represent projections of confocal Z-stacks. Asterisks denote plasma membrane protrusions, enriched in CD63 and LAMP-1, induced by depletion of rabip4s. Scale bar, 10 µm (C).