Figure 3. Phospho-Specific Antibodies Reveal Wg-regulated Segmental Expression Patterns of pMadMAPK and pMadGSK3.(A and B) Western blot analysis of pMadMAPK and pMadGSK3 antibodies demonstrating that they were phospho-specific, and that GSK3 phosphorylation had an absolute requirement for MAPK priming. Drosophila S2 cells were transiently transfected with the plasmids indicated. (C) Cultured 293T cells stably transfected with Mad-Flag treated with L-cell control conditioned medium (CMed), Wnt3a medium, control DMEM (Con), or 30 mM LiCl in DMEM for 2 hours. Wnt3a and LiCl inhibited the MadGSK3 phosphorylation band and increased β-Catenin levels (indicating that the Wnt treatment was effective). (D and E) pMadMAPK and pMadGSK3 antibodies stain the entire blastoderm and a Dpp-dependent dorsal stripe (inset). (F) pMadMAPK tracks ventral EGFR-activated MAPK (inset shows diphospho-Erk staining). (G and H) Segmental staining of pMadMAPK (Stage 9) and pMadGSK3 (Stage 17). (I) In Wg null mutants segmental expression is lost. Mutant embryos were identified by lack of staining with Wg antibody. Inset shows same embryo stained with DAPI to indicate that, despite its abnormal shape, it reached late stages of development. (J–L) Wg stabilizes pMadMAPK, overlapping with Wg stripes. (M–O) Nuclear pMadGSK3 accumulates in between Wg stripes, indicating that Wg inhibits Mad phosphorylation at GSK3 sites in vivo. (P–R) Wg overexpression driven with prd-Gal4 stabilizes pMadMAPK over a broader domain compared to just MWT alone (compare brackets in P and R). This experiment shows that Wg expression stabilizes pMADMAPK.
Image published in: Eivers E et al. (2009)
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