Figure 5. Corkscrew associates preferentially with and dephosphorylates Draper-II.

(a) HA-tagged Draper constructs and wild type Corkscrew (Csw^WT) were transfected into insect S2 cells. Anti-Csw immunoprecipitation (IP) samples were analyzed by anti-HA WB to compare Csw association with Draper-I and Draper-II. Anti-HA WB was performed on cell lysates to confirm Draper::HA transfection.

(b) Draper-II::HA was co-transfected with Csw^WT or Csw^CS. Anti-HA WB was performed on anti-Csw IP samples to compare Draper-II::HA association with Csw^WT versus Csw^CS. Blots were reprobed for Csw. Anti-HA WBs on lysates confirmed Draper-II::HA expression.

(c) Cells were transfected with indicated constructs and anti-HA IP was performed on cell lysates. Anti-phosphotyrosine and anti-HA WBs were performed on IP samples. Co-transfection of Csw^WT with Draper-II significantly reduced the tyrosine phosphorylation level of bands corresponding to the sizes of Shark and Draper-II (lane 7 versus 8). Csw^WT did not affect phosphorylation of Draper-I::HA or Shark in the absence of Draper-II::HA (lane 5 versus 6).

(d) Anti-phosphotyrosine, anti-HA, and anti-Myc WBs blots were performed on anti-HA IP samples from cells transfected with indicated constructs. Csw^CS increased the phosphorylation status of Shark and Draper-II when transfected together (lane 6 versus 7) but did not significantly alter Draper-I or Shark when expressed together in S2 cells (lanes 4 versus 5).

(e) Anti-Myc IP was performed on cells transfected with Draper-II::HA, Myc::Shark, and noted versions of Csw. Anti-phosphotyrosine, anti-HA, and anti-Myc WBs on IP samples are shown. Tyrosine phosphorylation of Shark was reduced by Csw^WT (lane 2 versus 3) but increased by Csw^CS (lane 2 versus 4).