Figure 2: Hh and Ihog can complex with the exosome marker CD63 in vivo.
From: Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion
(a) Lateral view of a reconstructed z-stack of confocal images from a wing disc expressing the exosome marker UAS.CD63-GFP and UAS.Ihog-RFP under the control of hh.Gal4/tubG80ts after 24 h at the restrictive temperature and immuno-labelled for Hh. Note the co-localization of Ihog-RFP (red) and the endogenous Hh (grey) with the marker CD63-GFP (arrow heads). Scale bar, 20 μm. (b) Live imaging of Drosophila abdomen shows CD63-GFP labelled puncta that co-localizes with puncta also labelled by Ihog-RFP (arrow heads) and that move along the cytoneme (See also Supplementary Movie 3). Scale bar, 10 μm. (c–e) Western blots showing co-immunoprecipation of CD63-GFP, Ihog-RFP and endogenous Hh after a GFP-Trap pull down (Chromoteck) from a high-speed supernatant of homogenized larvae expressing CD63-GFP as control and larvae co-expressing CD63-GFP and Ihog-RFP as expermiental. (c) Immunoprecipitation of CD63-GFP shown for both, the IP control and the experimental IP control (arrowhead). (d) Co-immunoprecipitation of Ihog-RFP revealed by anti-RFP in the experimental IP control (arrow) and not present in the IP control. (e) Co-immunoprecipitation of endogenous Hh revealed (after membrane antibody stripping) by anti-Hh in the experimental IP control (asterisk) and not present in the IP control, probably due to an enrichment effect of the co-expression of Ihog-RFP and CD63-GFP.
