Fig. 1.

Membrane attack complex-induced and endosome-dependent stabilization of ZFYVE21. Human umbilical vein endothelial cells (HUVECs) were treated with panel reactive antibody (PRA) sera (25% v/v in gelatin veronal buffer) (a). HUVECs were treated with MG132 (25 μM) (b). HUVECs were treated with IgG+ and IgG− sera fractions from PRA and C6-deficient sera (50% v/v in gelatin veronal buffer) for 30 min prior to western blotting (c, left). HUVECs were treated with IgG+ and IgG− sera fractions from PRA sera, IgG− sera fractions from C9-deficient sera, C9 AF647, or in combination for 30 min (c, right). HUVECs were pretreated with Dynasore (80 μM) or Pitstop2 (30 μM) for 30 min, d). HUVECs stably transduced with Rab5 wild type (WT) or Rab5 dominant negative (DN) (S43N) were treated with PRA prior to co-immunoprecipitation of Rab5 (e). Three-color confocal microscopy of PRA-treated (f, top scale bar 8 μm, bottom scale bar 530 nm). Recombinant ZFYVE21 (1 μg) was co-incubated with lipid-coated beads and probed by Western blot (g). HUVECs were treated for 30 min with KU-55933 prior to blotting of Rab5 co-immunoprecipitates (h) and whole-cell lysates (i). HUVECs stably transduced with control or MTM1-expressing vectors were treated with PRA sera for 30 min prior to Rab5 co-immunoprecipitation (j). Rab5+ZFVYE21+ endosomes were quantified by confocal microscopy (5 nM, k, top row, scale bar: 10 μm). Intermolecule distances between Rab5 and ZFYVE21 were calculated using super-resolution microscopy (k, bottom row, scale bar: 10 μm). Myc-tagged ZFYVE21 was used in far western blots (l, left blot). Far western blot membranes were stripped and probed by western blot (l, right three blots). Recombinant Rab5 (m, top), Rab5 WT, or Rab5 DN (m, bottom) were incubated with GDP or GTPγS prior to addition of ZFYVE21. HUVECs transfected with control or ZFYVE21 siRNA (n). HUVECs transfected with control of ZFYVE21 siRNA were treated with PRA for 4 h prior to immunofluorescence (I.F.) analysis (o, p, scale bar: 85 μm). Lipid reporter mean fluorescent intensity PI(3)P:PI(3,4,5)P3 ratios gated on C9+Rab5+ vesicles were calculated (q, r, scale bar: 10 μm). *p < 0.05, analysis of variance. For I.F., ≥3 fields were analyzed per group per experiment, and each experiment was repeated two times. For confocal microscopy analyses, ≥10 individual cells were analyzed per group, and each experiment was repeated four times. Super-resolution microscopy was repeated twice. All western blot assays were conducted two to four times. Representative data shown