Fig. 5. S458 is phosphorylated and is required for normal embryonic polarity. (A-C) Western blots of extracts from wild-type adult worms or worms expressing various GFP:MEX-5 fusion proteins as listed. Black triangles indicate 53 kDa (predicted size of endogenous MEX-5); white triangles indicate 82 kDa (predicted size of GFP:MEX-5). (A) Extracts from wild-type worms treated with alkaline phosphatase for 0, 2 or 14 hours and stained with either MEX-5 or MEX-5(pS458). (B) MEX-5(pS458) stains endogenous MEX-5 but not GFP:MEX-5^S458A for two independent lines of worms; compare with levels of GFP:MEX-5 stained by MEX-5. Note that the levels of all GFP:MEX-5 fusion proteins are much lower than endogenous MEX-5. (C) The GFP:MEX-5^{(KRRTSL to AAATSA)} fusion protein is stained by GFP, but not by MEX-5(pS458). (D-D'') Single, transgenic mex-5(zu199);mex-6(pk440) embryo expressing GFP:MEX-5^S458A and stained for DNA (DAPI, D) and immunostained for GFP (D') and PIE-1 (D''); embryo is at the one-cell stage and in metaphase of first mitosis.