IMAGE

Fig. S1

ID
ZDB-IMAGE-140106-2
Source
Figures for Moore et al., 2013
Image
Figure Caption

Fig. S1 Etv2 protein expression. (A) SDS–PAGE gel of HEK293T lysates transfected with CMV-driven expression vectors encoding myc-tagged zebrafish Etv2, Ets1a, Fli1a, Fli1b, or left untransfected (mock). Lysates from each sample were run on triplicate immunoblots, which were individually probed with Etv2 or Fli1b polyclonal antiserum, or a monoclonal against the myc-epitope (9E10). The Etv2 polyclonal serum recognizes a single band that is the same size as that recognized following immunodetection for the myc epitope. (B) Tg(fli1a:negfp)y7 embryos at 18 hpf injected with 5 ng of control or Etv2 MO followed by immunostaining using Etv2 polyclonal serum and Alexa-568 secondary. Etv2 antibody staining is clearly visible in embryos injected with control MO, but absent in embryos injected with 5 ng of an Etv2 translation blocking morpholino. (C) Top, camera lucida drawings of embryo at approximately 24 hpf. Bottom, immunostaining of an Tg(fli1a:egfp)y1 embryo with Etv2 polyclonal serum and alexa-568 secondary at 24 hpf. Faint Etv2 expression can be observed in many EGFP-positive cells within the caudal vein plexus, while strong Etv2 expression is apparent in a separate EGFP-negative population of cells (indicated by a white bracket). Etv2 expression is not detectable in the caudal aorta at this time point (red arrows). (D) Two photon micrographs of Tg(fli1a:negfp)y7 embryos immunostained with Etv2 (left panels) or Fli1b (right panels) polyclonal serum. Top panels are signal from Alexa-568 secondary antibody. Bottom panels are overlay of Alexa-568 and EGFP fluorescence. Images are higher magnification views of embryos shown in Fig. 1 G and I. Left, arrows indicate EGFP-positive endothelial nuclei that do not express Etv2; arrowheads indicate EGFP-negative/Etv2-positive cells within the dorsal aorta. Right, arrows indicate EGFP-positive endothelial nuclei that also express Fli1b; arrowheads denote EGFP-negative/Fli1b-positive blood cells circulating within the dorsal aorta. (E) Flat mounted embryos at 5 ss immunostained with Etv2 polyclonal serum. Embryos were injected with Etv2 cMO and left in the dark (no UV exposure) or illuminated at approximately 2 ss with UV and allowed to develop for 1.5 h (1.5 h post UV) prior to fixation and immunostaining.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

Reprinted from Developmental Biology, 384(1), Moore, J.C., Sheppard, S., Shestopalov, I.A., Chen, J.K., and Lawson, N., Post-transcriptional mechanisms contribute to Etv2 repression during vascular development, 128-40, Copyright (2013) with permission from Elsevier. Full text @ Dev. Biol.