Cyclin G physically interacts with ASX in vivo. (A) Schematic representation of Cyclin G full-length (CycG) and truncated forms (CycG 1 to 130 and CycG 130 to 566 corresponding to amino-acids 1 to 130 and 130 to 566, respectively). Grey box: Cyclin domain; red box: PEST sequence. (B-D) Cyclin G interacts with ASX-C (amino-acids 1139 to 1668) in S2 cells; black arrowheads indicate co-immunoprecipitation. FLAG-CycG (B) and FLAG-CycG 130–566 (D) co-immunoprecipitate with Myc-ASX-C. Note that FLAG-CycG 130 to 566 co-migrates with IgG heavy chains. (C) Myc-ASX-C co-immunoprecipitates with FLAG-CycG 1 to 130. Immunoprecipitations were performed with anti-Myc, anti-FLAG, or anti-HA (Mock) antibodies. Immunoprecipitated proteins were revealed by Western blot, using anti-Myc (top panel) or anti-FLAG antibodies (bottom panel). In (B) and (D), asterisks indicate IgG heavy chains. S: supernatant after immunoprecipitation; IP: protein G-agarose beads. Five percent of the input or supernatant and 50% of the immunoprecipitate were loaded onto the gels. (E) Myc-Cyclin G co-immunoprecipitates with endogenous ASX in da > Myc-CycG
ΔP third instar larvae. Immunoprecipitated proteins were revealed by Western blot, using anti-Myc antibody.