FIGURE 8.

CRK1-mediated phosphorylation of Sec31 is required for anterograde transport of the secreted protein. Cells harboring the Sec31 RNAi construct (A), the CRK1 RNAi construct (B), or the overexpression construct containing either wild-type Sec31, Sec31-7A, or Sec31-7D (C) were stably transfected with a construct encoding the secretory protein BiPNAVRG-HA9. After induction with tetracycline for 24 h (Sec31 RNAi and overexpression of wild-type and mutant Sec31 proteins) or 48 h (CRK1 RNAi), cells were washed with fresh medium, resuspended in fresh medium, and treated with cycloheximide (CHX) for 4 h. Samples from cells and the medium were collected at the indicated times. BiPNAVRG-HA9 was immunoprecipitated from cell lysate and culture medium by anti-HA antibody, fractionated by SDS-PAGE, and detected by anti-HA antibody. Band intensity was determined using the ImageJ software, plotted against the time of cycloheximide treatment, and presented below the Western blots. Blue lines indicate the BiPNAVRG-HA9 protein detected in cells (non-secreted protein), and red lines indicate the BiPNAVRG-HA9 protein detected in the medium (secreted protein). Error bars indicate S.D. calculated from three independent experiments.