![Characterization of the interaction of WDR91 with retromer components. (A) GST and GST-SNX27(PX) 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. (B) Co-IP of Flag-WDR91 with GFP-SNX27(PX) without or with VPS34-IN1 treatment, and co-IP of Flag-WDR91 with the PtdIns3P binding-deficient mutant GFP-SNX27(PX-RYKR/4A). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (C–E) Co-IP of Flag-WDR91 with SNX1-GFP. Schematic representation of SNX1 is shown in C. Co-IPs of Flag-WDR91 with SNX1-GFP and GFP-SNX1(PX) are shown in D and E, respectively. (F–H) Co-IP of Flag-WDR91 with SNX2-GFP. Schematic representation of SNX2 is shown in F. Co-IPs of Flag-WDR91 with SNX2-GFP and GFP-SNX2(PX) are shown in G and H, respectively. (I–K) Co-IP of Flag-WDR91 with SNX5-GFP. Schematic representation of SNX5 is shown in I. Co-IPs of Flag-WDR91 with SNX5-GFP and GFP-SNX5(PX) are shown in J and K, respectively. (L–N) Co-IP of Flag-WDR91 with GFP-SNX6. Schematic representation of SNX6 is shown in L. Co-IPs of Flag-WDR91 with GFP-SNX6 and GFP-SNX6(PX) are shown in M and N, respectively. (O–Q) Co-IP of Flag-WDR91 with GFP-SNX3. Schematic representation of SNX3 is shown in O. Co-IPs of Flag-WDR91 with GFP-SNX3 and GFP-SNX3(PX) are shown in P and Q, respectively. (R) GST, GST-SNX3, and the indicated GST-SNX-BAR proteins 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. (S) GST, GST-SNX3(PX), GST-SNX1(PX), GST-SNX2(PX), GST-SNX5(PX), and GST-SNX6(PX) 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. (T) Co-IP of GFP-tagged VPS35 and VPS35 carrying the PRLYL motif mutation (GFP-VPS35[RL/AP]) with Myc-VPS26. IPs were performed with GFP-trap beads, and precipitated proteins were immunoblotted using Myc and GFP antibodies. (U) Co-IP of Flag-WDR91 with GFP-VPS35 or GFP-VPS35(RL/AP). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. Source data are available for this figure: SourceData FS4.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/12/10.1083_jcb.202203013/2/jcb_202203013_figs4.png?Expires=1771196087&Signature=sW~BtrtaFdY2F4lJ3tk5UbB93BjYvD8p38nv~a~7NJwnzyKghu9UiLM4aazsAMq3fnB8e1DbFQSeLnS1EXolpe~yJdpGuAzwE2pUmW42l906NQmGejOfBZDyxhGCHwV26r0RVpFJ9QDPmmyT9g7JkPRY2-ahL8OKd30dmsIVkqdMr4EuZfVnb9hrXOtLMXKqyp-vos2gpADN-ylDT327sZkOzZ9UkcaTTbULTgv2cwkVtzvNBTEY79j6whBvdSqjjaAXKEwyMy4GPDxlrOK6DjftCGVSAcRwzXhphsV9Ov2qRxEagsTWwHyRsbMkRRWpFs1gmnGWY2cGPk4VcosJfA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Characterization of the interaction of WDR91 with retromer components. (A) GST and GST-SNX27(PX) 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. (B) Co-IP of Flag-WDR91 with GFP-SNX27(PX) without or with VPS34-IN1 treatment, and co-IP of Flag-WDR91 with the PtdIns3P binding-deficient mutant GFP-SNX27(PX-RYKR/4A). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. (C–E) Co-IP of Flag-WDR91 with SNX1-GFP. Schematic representation of SNX1 is shown in C. Co-IPs of Flag-WDR91 with SNX1-GFP and GFP-SNX1(PX) are shown in D and E, respectively. (F–H) Co-IP of Flag-WDR91 with SNX2-GFP. Schematic representation of SNX2 is shown in F. Co-IPs of Flag-WDR91 with SNX2-GFP and GFP-SNX2(PX) are shown in G and H, respectively. (I–K) Co-IP of Flag-WDR91 with SNX5-GFP. Schematic representation of SNX5 is shown in I. Co-IPs of Flag-WDR91 with SNX5-GFP and GFP-SNX5(PX) are shown in J and K, respectively. (L–N) Co-IP of Flag-WDR91 with GFP-SNX6. Schematic representation of SNX6 is shown in L. Co-IPs of Flag-WDR91 with GFP-SNX6 and GFP-SNX6(PX) are shown in M and N, respectively. (O–Q) Co-IP of Flag-WDR91 with GFP-SNX3. Schematic representation of SNX3 is shown in O. Co-IPs of Flag-WDR91 with GFP-SNX3 and GFP-SNX3(PX) are shown in P and Q, respectively. (R) GST, GST-SNX3, and the indicated GST-SNX-BAR proteins 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. (S) GST, GST-SNX3(PX), GST-SNX1(PX), GST-SNX2(PX), GST-SNX5(PX), and GST-SNX6(PX) 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. (T) Co-IP of GFP-tagged VPS35 and VPS35 carrying the PRLYL motif mutation (GFP-VPS35[RL/AP]) with Myc-VPS26. IPs were performed with GFP-trap beads, and precipitated proteins were immunoblotted using Myc and GFP antibodies. (U) Co-IP of Flag-WDR91 with GFP-VPS35 or GFP-VPS35(RL/AP). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. Source data are available for this figure: SourceData FS4.
