Fig. 4. GFP::Cdc27 and GFP::Cdc27^P304A can support healthy embryonic development in established stable p340 (GFP::Cdc27) and p341 (GFP::Cdc27^P304A) transgenic cdc27^L7123 rescue lines, but that the GFP::Cdc27^P304A,P456A double mutant fusion protein does not. (A) The endogenous Cdc27 protein or GFP::Cdc27 and GFP::Cdc27^P304A fusion proteins are indicated by arrows in lanes 1-3 respectively. Some (11%) of endogenous Cdc27 is still present in lanes 2 and 3 with the embryo samples collected from parental fly genotypes of gfp::cdc27/gfp::cdc27; cdc27^L7123/cdc27^L7123 and gfp::cdc27^P304A/gfp::cdc27^P304A; cdc27^L7123/cdc27^L7123 respectively. These flies carry a mutation in the cdc27gene on the third chromosome. The mutation is caused by a P-element insertion at 519 bp upstream of the ATG start site of cdc27 (http://flybase.bio.indiana.edu/.bin/fbidq.html?FBal0087027#FBrf0111595) (Huang and Raff, 2002). Actin was detected as a loading control. (B) Western blot of late third instar larvae brain samples: lane 1, W⁶⁷ control; lane 2, Cdc27^L7123:cdc27^L7123/cdc27^L7123 homozygous original mutant derived from the cdc27^L7123/TM6B Tb mutant fly line; lane 3, GFP::Cdc27^P304A,P456A the attempted rescue homozygous mutant (gfp::cdc27^P304A,P456A/gfp::cdc27^P304A,P456A; cdc27^L7123/cdc27^L7123) derived from the cross gfp::cdc27^P304A,P456A/gfp::cdc27^P304A,P456A; cdc27^L7123/TM6B Tb. Endogenous Cdc27 or GFP::Cdc27 was revealed by probing with anti-Drosophila Cdc27 antibody (top panel); cyclin A and cyclin B levels are shown in the middle panel using anti-Drosophila cyclin A or cyclin B antibodies respectively; Actin (bottom panel) was detected as a loading control. (C) Relative expression levels of endogenous Cdc27 or GFP::Cdc27 in third instar larvae brains from wild-type control samples (W⁶⁷), Cdc27^L7123 (cdc27^L7123/cdc27^L7123 homozygous mutant) and GFP::Cdc27^P304A,P456A (the attempted rescue homozygous mutant: gfp::cdc27^P304A,P456A/gfp::cdc27^P304A,P456A; cdc27^L7123/cdc27^L7123) were quantified from the western blot results present in the top panel of B in lanes 1-3 of the endogenous Cdc27 bands and the GFP:: Cdc27^P304A,P456A band. The endogenous Cdc27 band intensity in wild-type sample is set to 100% as the comparison.