Specificity of N. vectensis antibodies tested by pre-adsorption experiments. (A) Western blots of N. vectensis gastrula extracts using specific antibodies against NvaPKC (a′), NvLgl (b′), NvPar-1 (c′), and NvPar-6 (d′). Pre-adsorption of the antibodies with a tenfold excess of the antigen peptide resulted in the elimination of the staining of a single band for NvaPKC and NvLgl (a′ and b′, respectively) and the titration of anti-NvPar-1(c′). Arrowheads indicate the molecular weight in KD. A red arrow indicates the band recognized by the antibody for each protein. (B) Whole-mount immunohistochemistry pre-adsorption experiments show that the staining pattern was eliminated in early embryos when pre-incubated antibodies against NvaPKC and NvLgl with the respective peptide. (C) shows the negative control when the primary antibody was not added.