Xenbase Image ID: 149038

Figure 3. E-Syt2 Is Required In Vivo for FGF Receptor Endocytosis and Signaling via the Clathrin Pathway(A) E-Syt2 depletion inhibits FGF receptor endocytosis in vivo. Each blastomere of four cell embryos was injected with 6 pmol of Morpholino C (Mo-C) and 20 pg of Xenopus Myc-FGFR1 mRNA. ACs were then isolated and incubated with anti-Myc antibody to label surface receptors. After incubation with bFGF at 4°C or 22°C for the indicated times, ACs were subjected to indirect immunofluorescence labeling to reveal receptor endocytosis. The upper panels show typical images and the lower panel the statistical analysis of receptor internalization.(B) Embryos were injected and ACs were isolated and treated with bFGF at 22°C as in (A), before analysis of cell extracts for ERK and phospho-ERK levels by immunoblotting.(C) Dominant-negative Dynamin also inhibits FGFR1 endocytosis in vivo. ACs expressing Myc-FGFR1 alone or with Dynamin K44E (Dyn-K44E) were prepared and treated with bFGF and were analyzed for receptor endocytosis as in (A). In (A) and (C), “n” indicates the number of cells scored, scale bar in micrographs represents 30 μm, and error bars indicate the standard error.(D) Induction of Xbra in ACs requires FGFR1 endocytosis upstream of VRas. Embryos were injected with either CA-FGFR1 or VRas and DynK44E mRNAs and Xbra and ODC mRNA levels determined by RT-PCR.(E) ACs expressing CA-FGFR1 were also treated with clathrin or caveolin inhibitors, chlorpromazine and nystatin before RT-PCR analysis as in (D).In (D) and (E), +RT and –RT refer to control PCR analysis of whole embryo RNAs with and without reverse transcriptase.See also Figure S3. Image published in: Jean S et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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