Characterization of the interaction of WDR91 with Rab7. (A) Schematic depiction of WDR91 truncations and mutants. (B) Co-IP of Flag-Rab7 with GFP-tagged WDR91 truncations. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to Flag and GFP. (C) Purified GST and GST-Rab7 were immobilized on glutathione–Sepharose beads and incubated with 35S-labeled WDR91 proteins prepared by in vitro translation. After extensive washing, bound proteins were resolved by SDS-PAGE and viewed with autoradiography. (D) Co-IP of HA-Beclin1 with GFP-tagged WDR91 truncations. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to HA and GFP. (E) Co-IP of Flag-tagged WDR91, WDR91(1–405), and WDR91(392–747) with GFP-tagged Rab7(WT), Rab7(Q67L), and Rab7(T22N). IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to Flag and GFP. (F) Purified GST, GST-Rab7(Q67L), and GST-Rab7(T22N) immobilized on glutathione–Sepharose beads were incubated with GTP or GDP as indicated and then incubated with 35S-labeled WDR91 proteins prepared by in vitro translation. After extensive washing, bound proteins were resolved by SDS-PAGE and viewed with autoradiography. (G) Confocal images of the colocalization of Flag-WDR91, Flag-WDR91(3A), or Flag-WDR91(Δ5) with mCh-Rab7. Bars, 5 µm. (H) Co-IP of GFP-Rab7(Q67L) with Flag-tagged WDR91(WT), WDR91(3A), and WDR91(Δ5). IPs were performed with GFP-trap beads, and precipitated proteins were detected with the indicated antibodies. (I) Co-IP of HA-Beclin1 with Flag-tagged WDR91(WT), WDR91(3A), and WDR91(Δ5). IPs were performed with HA antibody, and precipitated proteins were detected with antibodies to HA and Flag.
