VPS33B(p.Gly131Glu) alters VPS33B interaction with Rab25 and Rab11a. (a, b) Representative mIMCD3 cells coexpressing wt YFP-VPS33B (a) or YFP-VPS33B(p.Gly131Glu) (b), RFP-VIPAR and CFP-Rab25. Scale bars = 10 μm. (c) Quantification of the colocalization between VPS33B constructs and Rab25 in transfected mIMCD3 cells. Data are mean ± standard deviation, unpaired t-test, **** P ≤ 0.0001. (d, e) Representative mIMCD3 cells coexpressing wt YFP-VPS33B (d) or YFP-VPS33B(p.Gly131Glu) (e), RFP-VIPAR and Myc-Rab11a, stained with an α-Myc antibody. Scale bars = 10 μm. (f) Quantification of the colocalization between VPS33B constructs and Rab11a in transfected mIMCD3 cells. Data are mean ± standard deviation, unpaired t-test, **** P ≤ 0.0001. (g) α-HA co-IP on lysates of HEK293 cells coexpressing HA-VPS33B or HA-VPS33B(p.Gly131Glu) with Myc-VIPAR and Rab25-GFP. (h) a-HA co-IP on lysates of HEK293 cells coexpressing HA-VPS33B or HA-VPS33B(p.Gly131Glu) with Myc-VIPAR and Rab11a-GFP. CFP, cyan fluorescent protein; co-IP, co-immunoprecipitation; HA, hemagglutinin; HEK, human embryonic kidney; GFP, green fluorescent protein; IP, immunoprecipitation; mIMCD3, murine inner medullary collecting duct 3; RFP, red fluorescent protein; wt, wild type; YFP, yellow fluorescent protein.