Fwd binds and colocalizes with the recycling endosome regulator Rab11. (A) Yeast two-hybrid assays. (Left) Patches of yeast cells cotransformed with Fwd or FwdKD bait plasmids together with one of several prey plasmids (vector alone, Rab5, Rab7, Rab11, Rab11Q70L, and Rab11S25N). (Right) Filter X-gal assays performed on replicates of these patches. Blue color indicates a positive interaction. (B) Quantification of yeast two-hybrid results. LacZ reporter expression (β-galactosidase units) induced by various combinations of bait and prey plasmids (as in A). Statistically significant differences are: (single asterisk) P < 0.001, (double asterisk) P < 0.05, (triple asterisk) P < 0.01. (C) Co-immunoprecipitation (coIP) of Rab11 with Fwd and FwdKD expressed in COS-7 cells. Immunoblots were probed with anti-HA or anti-Flag, as indicated on the right. IP with anti-Flag antibody pulls down HA-Fwd and HA-FwdKD only when coexpressed with Flag-Rab11 (top panels). IP with anti-HA pulls down Flag-Rab11 only when coexpressed with HA-Fwd or HA-FwdKD (bottom panels). Note that a nonspecific protein of slightly lower mobility than Flag-Rab11 is present in all lanes of the anti-HA IP experiment. (D) Phase–contrast (phase) and corresponding fluorescence micrographs of spermatocytes coexpressing YFP-Rab11 and CFP-Fwd. Colocalization (arrows) of YFP-Rab11 (magenta) and CFP-Fwd (green) appears white (overlay). Bar, 20 µm.
