Figure 5.
WDR91 promotes Rab7 interaction with SNX-retromer components. (A) Co-IP of Flag-WDR91 with GFP-SNX27. IP was performed with anti-Flag antibody resin and the precipitated proteins were detected with Flag and GFP antibodies. (B) Schematic depiction of SNX27 truncations. (C–E) Co-IP of Flag-WDR91 with GFP-tagged PDZ (C), PX (D), and FERM (E) of SNX27. IPs were performed with anti-Flag antibody resin and detected with Flag and GFP antibodies. (F) Schematic depiction of WDR91 truncations. (G) Co-IP of Flag-tagged WDR91(1-405) and WDR91(392-747) with GFP-SNX27(PX). IPs were performed with anti-Flag antibody resin and detected with Flag and GFP antibodies. (H) GST and GST-SNX27 immobilized on glutathione-Sepharose beads were incubated with WDR91-His6. The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (I) GST and GST-SNX27(PX) immobilized on glutathione-Sepharose beads were incubated with His6-WDR91(392-747). The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (J) Co-IP of GFP-tagged Rab7(WT), Rab7(Q67L), and Rab7(T22N) with Myc-SNX27. IPs were performed with GFP-trap beads, and precipitated proteins were detected with Myc and GFP antibodies. (K) Co-IP of GFP-Rab7(Q67L) with Myc-SNX27 without or with Flag-WDR91. IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc, and GFP antibodies. (L) Co-IP of Flag-WDR91(WT), Flag-WDR91(3A), and Flag-WDR91(Δ5) with GFP- SNX27(PX). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (M) Co-IP of Myc-SNX27 with GFP-Rab7(Q67L) or GFP-Rab7(T22N) in the presence of Flag-WDR91(WT), Flag-WDR91(3A), or Flag-WDR91(Δ5). IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc and GFP antibodies. (N) Co-IP of Flag-WDR91 with GFP-VPS35. IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (O) GST and GST-VPS35 immobilized on glutathione-Sepharose beads were incubated with WDR91-His6. The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (P) Co-IP of GFP-Rab7(Q67L) with Myc-VPS35 in the presence of Flag-WDR91. IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc, and GFP antibodies. (Q) Co-IP of endogenous VPS35, SNX27, and WDR91 with ectopically expressed GFP-Rab7(Q67L) in control and KO-91 HeLa cells. Proteins were precipitated with GFP-trap beads and detected with antibodies against GFP, WDR91, VPS35, and SNX27. (R) Co-IP of endogenous VPS35, SNX27, SNX6, SNX3, and Rab7 with WDR91 in control HeLa cells. IPs were performed with WDR91 antibody and precipitated proteins were detected with antibodies against the indicated proteins. (S) Co-IP of endogenous WDR91, VPS35, SNX27, SNX6, and SNX3 with Rab7 in Ctrl cells, KO-91 cells, and KO-91 cells transfected with empty or Flag-WDR91–expressing vectors. IPs were performed with Rab7 antibody and precipitated proteins were detected with antibodies against the indicated proteins. Source data are available for this figure: SourceData F5.

WDR91 promotes Rab7 interaction with SNX-retromer components. (A) Co-IP of Flag-WDR91 with GFP-SNX27. IP was performed with anti-Flag antibody resin and the precipitated proteins were detected with Flag and GFP antibodies. (B) Schematic depiction of SNX27 truncations. (C–E) Co-IP of Flag-WDR91 with GFP-tagged PDZ (C), PX (D), and FERM (E) of SNX27. IPs were performed with anti-Flag antibody resin and detected with Flag and GFP antibodies. (F) Schematic depiction of WDR91 truncations. (G) Co-IP of Flag-tagged WDR91(1-405) and WDR91(392-747) with GFP-SNX27(PX). IPs were performed with anti-Flag antibody resin and detected with Flag and GFP antibodies. (H) GST and GST-SNX27 immobilized on glutathione-Sepharose beads were incubated with WDR91-His6. The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (I) GST and GST-SNX27(PX) immobilized on glutathione-Sepharose beads were incubated with His6-WDR91(392-747). The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (J) Co-IP of GFP-tagged Rab7(WT), Rab7(Q67L), and Rab7(T22N) with Myc-SNX27. IPs were performed with GFP-trap beads, and precipitated proteins were detected with Myc and GFP antibodies. (K) Co-IP of GFP-Rab7(Q67L) with Myc-SNX27 without or with Flag-WDR91. IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc, and GFP antibodies. (L) Co-IP of Flag-WDR91(WT), Flag-WDR91(3A), and Flag-WDR91(Δ5) with GFP- SNX27(PX). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (M) Co-IP of Myc-SNX27 with GFP-Rab7(Q67L) or GFP-Rab7(T22N) in the presence of Flag-WDR91(WT), Flag-WDR91(3A), or Flag-WDR91(Δ5). IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc and GFP antibodies. (N) Co-IP of Flag-WDR91 with GFP-VPS35. IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (O) GST and GST-VPS35 immobilized on glutathione-Sepharose beads were incubated with WDR91-His6. The precipitates were immunoblotted with His6 antibody. GST-fusion proteins were stained with Coomassie blue. (P) Co-IP of GFP-Rab7(Q67L) with Myc-VPS35 in the presence of Flag-WDR91. IPs were performed with GFP-trap beads and immunoblotted using Flag, Myc, and GFP antibodies. (Q) Co-IP of endogenous VPS35, SNX27, and WDR91 with ectopically expressed GFP-Rab7(Q67L) in control and KO-91 HeLa cells. Proteins were precipitated with GFP-trap beads and detected with antibodies against GFP, WDR91, VPS35, and SNX27. (R) Co-IP of endogenous VPS35, SNX27, SNX6, SNX3, and Rab7 with WDR91 in control HeLa cells. IPs were performed with WDR91 antibody and precipitated proteins were detected with antibodies against the indicated proteins. (S) Co-IP of endogenous WDR91, VPS35, SNX27, SNX6, and SNX3 with Rab7 in Ctrl cells, KO-91 cells, and KO-91 cells transfected with empty or Flag-WDR91–expressing vectors. IPs were performed with Rab7 antibody and precipitated proteins were detected with antibodies against the indicated proteins. Source data are available for this figure: SourceData F5.

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