Fig. 7. UNC-51-dependent phosphorylation of UNC-14 and VAB-8. (A,B) Western blots were probed with anti-HA antibodies to detect UNC-14. (A) COS cell extracts expressing UNC-14 alone, or with wild-type or mutant UNC-51 proteins. UNC-14 was phosphorylated when coexpressed with wild-type UNC-51, partially phosphorylated when coexpressed with UNC-51(K39R) and not phosphorylated when coexpressed with UNC-51(AIKAI). (B) All lanes contained UNC-14 that was immunoprecipitated from COS cells expressing both wild-type UNC-51 and UNC-14. The sample from the second lane was treated with phosphatase ( PPase) to yield a lower molecular mass band that ran at the same size as the product from cells expressing only UNC-14. Adding the phosphatase inhibitors Na₃VO₄ or EDTA inhibited the ability of PPase to yield the lower molecular mass UNC-14 band. (C) GST and GST-UNC-14 proteins were expressed and purified from E. coli, and incubated with [-³²P]ATP and wild-type UNC-51 purified from COS cells. GST-UNC-14 was labeled, whereas no labeling was seen with the GST control. (D,E) Western blots were probed with anti-Myc antibodies to detect VAB-8. (D) COS cell extracts expressing VAB-8 alone, or with wild-type or mutant UNC-51 proteins. VAB-8 was phosphorylated when coexpressed with wild-type UNC-51 or with UNC-51(K39R), but not phosphorylated when coexpressed with UNC-51(AIKAI). (E) All lanes contained VAB-8 that was immunoprecipitated from COS cells that expressed both wild-type UNC-51 and VAB-8. PPase treatment of the sample in the second lane converted multiple bands into one lower molecular mass band that ran at the same size as the product from cells expressing only VAB-8. Adding the phosphatase inhibitors Na₃VO₄ or EDTA inhibited the ability of PPase to yield the lower molecular mass VAB-8 band.