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Blood, Vol. 111, Issue 3, 1157-1166, February 1, 2008
Fibroblast growth factor controls the timing of Scl, Lmo2, and Runx1 expression during embryonic blood development
Blood Walmsley et al.
111: 1157
Supplemental materials for: Walmsley et al
Files in this Data Supplement:
- Figure S1. SU5402 blocks FGF/MAPK signaling in a dose dependent manner (PDF, 34.8 KB) -
Stage 8 embryos were treated with 25µM (lane2) 50µM (lane3) or 100µM (lane4) SU5402 in 0.1XMBS and grown to stage10.5 when they were snap frozen in dry ice and processed for Western blotting. Control embryos (lane1) were cultured in 0.1XMBS containing 1% DMSO. Protein extracts were probed for pMAPK (mouse anti-pERK 1:3000) followed by  -actin (mouse anti -actin 1:1000) as a loading control.
- Figure S2. Dose dependent effects of activin mRNA on the morphology of animal caps (PDF, 55.5 KB) -
Embryos at the one cell stage were injected in the animal pole with 200, 400, 800 or 1200 fg activinB mRNA (B-E) and grown to stage 8 when animal caps were excised along with uninjected control caps (A). Caps were cultured to neurula stages (as judged by sibling embryos) and their morphology was examined. Optimal elongation representing dorsal mesoderm occurs at 400-800fg activin. At higher levels of activin endoderm starts to be induced (asterisk in E).
- Figure S3 (PDF, 47.8 KB) -
(A) Dose dependent effects of XFD mRNAs on the morphology of activin induced animal caps. Single-cell embryos were injected in the animal pole with 400fg activin mRNA (B within subset) or 400fg activin mRNA plus 200, 400 and 800pg XFD mRNA (C-E). Caps were excised at stage 8 and cultured alongside uninjected control caps (A within subset) until sibling embryos reached stage 16. Some caps were harvested and snap frozen in dry ice at stage 10.5 for Western blot analysis (see Fig. S3B). Activin elongates the caps (B within subset) but this elongation is blocked by injection of 200-800pg XFD. (C-E within subset). (B) Dose dependent block of activin induced FGF/MAPK signaling in animal caps by the dominant negative FGF receptor XFD. Animal caps from embryos treated as in Fig S3A were cultured to stage 10.5 then processed for Western blotting. Protein extracts were probed for pMAPK (mouse anti-Perk 1:3000) followed by -actin (mouse anti- actin 1:1000) as a loading control. Activin induces pMAPK (compare lane 2 with uninjected caps in lane1) and this is blocked by 200-800pg of XFD in a dose dependent manner (lanes 3-5, 200, 400 800 pg XFD mRNA respectively). Lane 6, negative control using stage 5 embryos and lane 7 positive control, stage 10.5 whole embryos.
Western Blotting
Protein extraction and Western blot analysis were as described 28, except that 10% Bis-Tris gels were used. pMAPK antibody (clone MAPK-YT), anti-mouse IgG peroxidase and mouse anti--actin (clone AC-15) from Sigma were diluted 1:3000, 1:4000 and 1:1000 respectively in 0.5% Marvel/PBSTw.
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