FIGURE 3:
C-terminal Cactus fragments are generated by the action of CalpA and are present in Dl complexes. (A) C-terminal, GFP-tagged CalpA and Cact constructs are visualized in 0- to 2-h-old embryos and in S2 cells by Western blot. Arrowheads indicate full-length tagged proteins (125, 90, 85, and 65 kDa for CalpA-eGFP, Cact-eGFP, CactΔPEST-eGFP, and CactE10-eGFP respectively), and arrows indicate C-terminal fragments (a for Cact-eGFP, b for CactΔPEST-eGFP fragments). Note that the Cact-eGFP fragment migrates slightly faster than CactE10-eGFP. Asterisk denotes nonspecific band detected by the anti-GFP antibody. Tubulin was used as loading control. (B) Analysis of Cact-eGFP protein over time. Embryos containing six copies of maternally driven Cact-eGFP constructs were collected at 2-h intervals. Full-length Cact-eGFP (90 kDa) decreases over time at a faster rate than C-terminal fragments (64 kDa). Bars represent Cact-eGFP relative to tubulin protein levels. Statistical significance defined by Student's t test, *p < 0.05. (C) CalpA generates Cact fragments. To attain high levels of Cact-eGFP expression, S2 cells were transfected with pT>cact-eGFP, Actin-Gal4, and pMT>CalpA-V5/His. After 24 h in the presence of CuSO4 to induce CalpA expression, cells were treated 5 h with EGTA, subsequently washed with fresh medium for the times indicated, and submitted to immunoprecipitation for anti-GFP. Arrowhead indicates full-length Cact-eGFP, which decreases over time (GFP IP, left), whereas the amount of a Cact-eGFP fragment (arrow) increases. Equivalent amounts of the same samples submitted to GFP IP were used for IP with anti-Dl (Dl IP, right). Both full-length and Cact-eGFP fragments coimmunoprecipitate with Dl. The load portion of the anti-V5 blot was underexposed to reveal that the decrease in CalpA-V5 in the GFP IP lanes after 1 h EGTA wash is not due to a smaller protein input for IP. (D) Lysates from wild-type (WT) embryos or embryos expressing six copies of cact-eGFP were immunoprecipitated with anti-Dl. Endogenous Cact (Cactus), as well as Cact-eGFP (GFP) full-length (arrowhead) and C-terminal fragments (arrow), coimmunoprecipitate with Dl.