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Fig. 6. Casanova protein is phosphorylated by MAPK in vivo. (A)
Putative conserved MAPK phosphorylation sites (red box) in Casanova and in
Sox17 from human, mouse and zebrafish. The blue line corresponds to the 15
amino acid peptide used to generate antibodies (`
Casanova-Phospho'
antibody). (B-F) Western blot of protein extracts of embryos at 30%
epiboly (15 embryos per lane). Embryos were injected with cas-flag
RNA at 100 ng/µl into one blastomere at the two-cell stage. Zebrafish
mkp3 RNA (50 ng/µl) (D) or SB203580 (200 µM) (F) were
co-injected with cas-flag RNA, while 15 µM SU5402 was added to the
medium after the injection (E). Overexpressed Casanova proteins are Flag
tagged and detected by a mouse anti-Flag antibody. (B) The `
Casanova-Phospho' antibody recognised the product of ectopically expressed
Casanova RNA (lane 2). Product of casanova/sox32-S47A form RNA is not
recognised by the antibodies directed against the phosphorylated consensus
site (lane 3). (C-E) The level of phosphorylated overexpressed Casanova
increases following overexpression of an activated form of ERK (C, compare
lane 1 with 2) but decreases following inhibition of FGF-MAPK signalling
(D,E). It is not affected by the P38-MAPK inhibitor that has been shown to
work in zebrafish (F) (Fujii et al.,
2000).
