Pax6 and AR interact directly in vivo.
(A–C) FRET between Pax6 and AR. HeLa cells transiently expressing a 1∶1 ratio of CFP-AR (pDestECFP-AR) and YFP-Pax6 (pDestPax6-EYFP) were subjected to FRET as described in the methods section. A schematic illustration of CFP-AR and YFP-Pax6 before and after acceptor photobleaching is presented in A. The cell images in B are visualized in pseudo-colors before and after acceptor photobleaching. The YFP acceptor was bleached, and FRET detected as decreased YFP-emission at 532 nm and a corresponding increased CFP-emission at 479 nm in the bleached area. The graphical display in B shows the emission spectrum of CFP-AR together with YFP-Pax6 at 479 and 532 nm, respectively, before (blue) and after (red) acceptor photobleaching. (C) Percent FRET between CFP-AR and Pax6-YFP compared with FRET between CFP-YFP, CFP-Pax6-YFP, and CFP-Pax6 - Pax6-YFP. The FRET efficiency was calculated as described in the methods section. Each bar represents the mean of 3–5 experiments. (D) Coimmunoprecipitation of GFP-AR and 3×Flag-Pax6. HeLa FlpIn 3×Flag-Pax6 cells were induced to express 3×Flag-Pax6 and subsequently transfected with pDestEGFP-AR or pEGFP-C1 using Metafectene Pro (Biontex). A GFP-antibody (Abcam) was used to immunoprecipitate GFP-AR-3×Flag-Pax6 complexes from the cells. The upper right gel shows the 0.5% input of 3×Flag-Pax6 and the lower right gel the 0.5% input of GFP and GFP-AR. The upper left gel shows that 3×Flag-Pax6 was immunoprecipitated together with GFP-AR, but not with the GFP control.