Figure 9.
Delineation of functional domains in hVam6p. (A) Schematic representation of full-length hVam6p and various deletion constructs used in these experiments. (B) HeLa cells were cotranfected with plasmids encoding Myc– and HA–hVam6p, or hVam6p deletion mutants, or a control epitope-tagged protein (HA–JNK1), as indicated. After 18 h, cells were labeled for 8 h with [35S]methionine, detergent extracted, and subjected to immunoprecipitation–recapture with the antibodies indicated. (C-N) HeLa cells were transfected with plasmids encoding Myc– or HA–Vam6p full-length or deletion constructs. After 24 h, fixed-permeabilized cells were coincubated with rabbit polyclonal antibodies to either the Myc or HA epitopes together with mouse monoclonal antibody to lamp-1. Bound antibodies were revealed by Alexa-488–conjugated donkey anti–mouse antibody (green channel) and Cy3-conjugated donkey anti–rabbit IgG (red channel). The third panel in each row was generated by merging of the images in the red and green channels. Arrows mark the coalescence of lysosomes into juxtanuclear regions. Bar, 10 μm.