Fig. 2. Localization and half life of QNR-71 in cells from the quail retinal pigmented epithelium. VSV-G-tagged QNR-71 was transiently expressed in quail cells dissociated from the retinal pigmented epithelium at E8. The cells were then fixed and processed for immunofluorescence using the monoclonal P5D4 anti-VSV-G antibody (a,d). (b) The phase contrast image of a, where pigment granules appear as black rod structures. In (c), pigmented quail or HeLa cells were grown on 24-well dishes and transfected with DNAs encoding VSV-G-epitope tagged QNR-71 as indicated in Materials and Methods. The cells were then pulse labeled for 30 minutes with [³⁵S]methionine/cysteine and chased for the indicated period of time. QNR-71 was immunoprecipitated with the anti-VSV-G antibody, and analyzed by SDS-PAGE. The position of the precursor (P) and mature (M) forms of the immunoprecipitated proteins are indicated. (d) Cells were first treated with 10 mM NH₄Cl for 1 hour before fixation. (d) is the superimposition of phase contrast and fluorescence images. Some of the large vacuoles contain both QNR-71 and melanin pigments.