FIGURE 7.

GPR107 is dispensable for anterograde protein transport and for maintaining the morphological structure of the trans-Golgi network. A, equal number of wild type and GPR107-deficient KBM7 cells were labeled with [³⁵S]methionine/cysteine for 10 min and chased at 37 °C or kept on ice. Supernatants were collected at the indicated time points and analyzed by SDS-PAGE and autoradiography. B, wild type and cells lacking GPR107 were pulse-labeled with [³⁵S]methionine/cysteine as in A and chased for the indicated time points. Cells were lysed, and class I MHC molecules were recovered using W6/32 antibody, treated with Endo H, and analyzed by SDS-PAGE and autoradiography. C, KBM7^WT and GPR107^GT expressing C-terminally HA-tagged furin were labeled with [³⁵S]methionine/cysteine for 20 min and chased for different time points. Both the cells and the supernatants were collected in parallel, lysed, and immunoprecipitated with anti-HA-coupled beads and where indicated subjected to PNGase F treatment and then analyzed by SDS-PAGE and autoradiography. furin_i is intracellular furin; furin_s is secreted furin. D, HeLa cells expressing HA-tagged GPR107 were transduced with the control shGFP or two sets of shRNA that targets GPR107. Cell lysates were prepared from these samples, and knockdown efficiency was examined by Western blotting using anti-HA HRP. E, confocal images of control and GPR107-depleted HeLa cells before or at different time points after BFA treatment. Cells were stained for the trans-Golgi network marker TGN46 or furin (left panel). In all images, the nuclei were stained with DAPI (blue).