Fig. 4. aPKC protein but not its mRNA localizes to the CAB. (A) aPKC antibody recognizes a single 65 kDa protein in Phallusia mammillata egg extracts (lane 1). The signal disappears when aPKC antibody is pre-incubated with the specific antigenic peptide (lane 2) but not with an unrelated peptide (lane 3). (B) Immunoblot of protein extracts from different developmental stages (E, egg; 2, 2 cells; 8, 8 cells; 16, 16 cells; 32, 32 cells; G, gastrula; N, neurula; T, tadpole). (C-J) Confocal images of Phallusia embryos immunolabelled for aPKC protein. Arrowhead indicates the pair of CABs. Stages can be compared with drawings in Fig. 1 and posterior vegetal blastomere name is indicated (B4.1, B5.2, B6.3, B7.6). Each image represents a projection of two to four confocal sections containing the brightest aPKC label. (C) 4-cell stage, vegetal view. (D) 8-cell stage, posterior surface view. (E) 8-cell stage, lateral view; the two halves of the embryo are projected into one plane so that both left and right CABs are visible. Inset shows a drawing of the orientation of the embryo as depicted in Fig. 1B. (F) 16-cell stage, vegetal view. (G) 32-cell stage, vegetal view. (H) 64-cell stage, vegetal view. (I) 8-cell stage, high magnification posterior surface view. Movie 2 in supplementary material shows the complete series of confocal z sections corresponding to this image. (J) B5.2 blastomeres from two 16-cell stage embryos showing cell-cycle-dependent compaction of the CAB. (K-N) aPKC mRNA is maternal and ubiquitously distributed. (K) Northern blot. aPKC probe was hybridized to RNA prepared from Phallusia embryos at the indicated stages (E, unfertilized egg; 2: 2 cells; 8: 8 cells; 32: 32 cells; G: gastrula; N, neurula; T, tadpole). (L-N) In situ hybridization using aPKC antisense probe (L,N) or negative control sense probe (M). The CAB is visible as in most fixed embryos (arrowhead in N) but not labelled by the probe for aPKC mRNA. Bars, 15 µm.