WDR91 is recruited to endosomes by interacting with GTP-Rab7. (A) Coimmunostaining of endogenous WDR91 with EEA1, Rab7, LAMP1, and Sec61-β in HeLa cells. (B) Quantification of the colocalization as shown in A. (C) Co-IP of Flag-WDR91 with GFP-tagged Rab7, Rab5, and Rab9. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to Flag or GFP. (D and E) Colocalization (D) and quantification (E) of endogenous WDR91 with Rab9 in cells treated with siCtrl and Rab7 siRNA (siRab7). (F) Immunoblotting of Rab7 in HeLa cells treated with siCtrl or siRab7. (G) Co-IP of GFP-tagged Rab7, Rab7(Q67L), and Rab7(T22N) with Flag-WDR91. IPs were performed with GFP-trap beads, and precipitated proteins were detected with Flag antibody. (H) Purified GST, GST-Rab7, GST-Rab7(Q67L), and GST-Rab7(T22N) were first incubated with GTP or GDP as indicated, immobilized on glutathione–Sepharose beads and then incubated with ³⁵S-labeled WDR91 prepared by in vitro translation. After extensive washing, bound proteins were resolved by SDS-PAGE and viewed with autoradiography. (I) Colocalization of Flag-WDR91 with mCh-CD63 in HeLa cells coexpressing BFP-Rab7, BFP-Rab7(Q67L), and BFP-Rab7(T22N). Zoom images represent magnified views of boxed areas. Bars, 5 µm. On graphs showing quantification of protein colocalization (B and E), Pearson’s correlation coefficient is plotted on the y-axis. ≥50 cells were scored. Error bars represent SEM. ***, P < 0.001.