Fig. 3.

RC-APEX baits biotinylate distinct proteins in live egg chambers. (A-F) Ovaries expressing indicated RC-APEX baits were permeabilized and biotinylated, then egg chambers were fixed and stained with TRITC-Phalloidin (A″-F″) and streptavidin-AF488 (A′-F′) to visualize F-actin and biotin. Representative images are shown of stage 6 or 7 egg chambers. Note the specific biotin signal at RCs for the RC-APEX fusions (A′-D′), the unlocalized cytoplasmic biotin signal for GBP::APEX (E′) and the absence of signal in the no-APEX control (F′). (A′) Yellow arrows denote biotinylated follicle cell RCs in (BAC) pav::GFP. (G-K) APEX-labeled ovaries were lysed in denaturing conditions, biotinylated proteins were captured by magnetic streptavidin beads, and eluates were analyzed by western blotting. (G) Inputs, unbound fractions and eluates were analyzed by western blotting with streptavidin-HRP to confirm that biotinylated proteins were present. (H-K) Western blot analysis was performed on the APEX eluates (labeled on top of lanes) to check for known RC proteins. (H) Pav::APEX protein was abundant in the Pav::APEX eluate. (I) APEX::Kelch was enriched in the APEX::Kelch eluate, whereas a background endogenous Kelch protein band could be detected in all other samples. (J) HtsRC::APEX and endogenous HtsRC were present in the HtsRC::APEX sample, whereas endogenous HtsRC was highly enriched in the APEX::KREP sample. (K) Filamin was observed only in the APEX::KREP sample. Scale bar: 50 μm.