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WDR44 patient variants enhance binding to RAB11 and cause RAB11 redistribution in cells. a Coimmunoprecipitation analysis of coexpressed GFP-RAB11A and Myc-WDR44 wild-type or variant in 293T for 48 h. Myc-tagged proteins were immunoprecipitated from 293T lysate and probed with GFP, Myc, and GAPDH. The plot (below) shows the relative co-immunoprecipitated levels of GFP-RAB11A bound to Myc-WDR44 wild-type or variants from three independent experiments. Statistical analysis comparing wild-type (WT), R733* and N840S are shown vs WT: P = 0.0269 (L668S), 0.0459 (R733*), 0.0236 (S764F), 0.0226 (G782C), 0.0261 (N840S), 0.0002 (D648G), 0.0009 (D669N), 0.0002 (H839R); vs R733*: P = 0.0459 (WT), 0.0221 (D648G), 0.0448 (S764F); vs N840S: P = 0.0261 (WT), 0.0333 (S764F), 0.0356 (G782C), 0.0018 (D669N), 0.0011 (H839R), 0.0004 (D648G). b Immunoprecipitation analysis of GFP-WDR44 wild-type or variants transiently expressed in RPE1 WDR44 KO F4 cells for 48 h. GFP proteins were immunoprecipitated from lysate and probed with GFP, RAB11, VAPA, and GAPDH antibodies. Blots are represented from three independent experiments. The plots (below) shows the relative co-immunoprecipitated levels of RAB11 and VAPA bound to GFP-WDR44 wild-type or variants. RAB11: P = 0.0183 (G782C), 0.0135 (N840S), 0.0086 (D648G), 0.0022 (L668S), 0.0065 (D669N), 0.0023 (R733*), 0.0005 (S764F), 0.0009 (H839R); VAPA: P = 0.0229 (R733*), P = 0.0087 (D648G). c IFM images of RPE1 WDR44 KO F4 cells transfected with GFP or GFP-WDR44 wild-type or variants. Cells express BFP and are immunostained with RAB11. Plasma membrane and nuclei are indicated by solid and dotted lines, respectively. Representative images from 20–25 cells are shown. White asterisk denotes untransfected cells. Scale bars, 10 μm. Mean ± s.e.m. from three independent experiments. Unpaired two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file. WCL whole cell lysate.
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