SNX1 is less efficiently recruited to CI-MPR tubules expressing a chimera harboring the WLM-AAA mutation. (A) Scheme of CI-MPR chimera constructs used. SP, signal peptide; TM, transmembrane domain. (B, top) The GFP–CI-MPR chimera WT and the GFP–CI-MPR chimera WLM-AAA mutant have comparable expression levels. HeLa cells were transfected with CI-MPR chimeras. 48 h after transfection, GFP levels were analyzed by Western blotting. (B, bottom) n = 3 independent experiments. (C) The GFP–CI-MPR chimera WLM-AAA mutant has a reduced ability to bind to the SNX1/2–SNX5/6 complex. GFP trap of GFP-tagged GFP–CI-MPR chimeras, each transiently transfected in HEK293T cells. Molecular masses are given in kilodaltons. IP, immunoprecipitation. (D, left) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR chimera constructs. Bars: (main images) 20 µm; (insets) 5 µm. (D, right) The percentages of cells with at least one GFP-positive tubule were blindly scored. n = 3 independent experiments; WT, 145 cells; WLM-AAA, 139 cells. (E) HeLa cells were transfected with WT or WLM-AAA mutant GFP–CI-MPR construct and immunostained for endogenous SNX1 and endogenous VPS35 after 48 h. Bars: (main images) 20 µm; (insets) 10 µm. (E, top right) Relative number of GFP-positive and SNX1-negative tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells. (E, bottom right) Relative number of GFP-positive and SNX1-positive tubules per cell. n = 3 blindly scored independent experiments; WT, 40 cells; WLM-AAA, 38 cells (means ± SEM; unpaired t test; *, P < 0.05; ***, P < 0.001). The total number of tubules analyzed in WT cells was 254 tubules and in WLM-AAA cells was 199 tubules.