Fig. 2.

ZFYVE21 modulates PI(3,4,5)P3 by vesicular removal of PTEN. Spectral counts of PTEN in vesicular proteomes (a). Human umbilical vein endothelial cells (HUVECs) transfected with control or PTEN siRNA were treated with panel reactive antibody (PRA) sera for 30 min prior to Rab5 co-immunoprecipitation and western blotting (b, left) or with PRA sera for 4 h prior to quantitative reverse transcriptase PCR (qRT-PCR) analysis (b, right). HUVECs stably transduced with Rab5 WT or Rab5 DN constructs were treated with PRA sera for 30 min prior to Rab5 co-immunoprecipitation (c, left) or confocal microscopic staining (c, right, left scale bar: 10 μm, right scale bar: 342 nm). HUVECs were treated with PRA for 30 min prior to downstream analyses by Rab5 co-immunoprecipitation (d), confocal microscopic staining (e, left scale bar: 10 μm, right scale bar: 251 nm), and enzymatic assays assessing vesicular PTEN activity (f). Co-immunoprecipitation studies were performed as indicated (g–i). *p < 0.05, analysis of variance. Proteomic analyses were performed three times using three separate HUVEC donors. qRT-PCR analyses were performed using technical triplicates and was repeated two times. *p < 0.05, Student’s t test. PTEN enzymatic activity assays utilized technical replicates and was repeated two times. Confocal microscopic analyses analyzed ≥5 cells per experiment, and each experiment was repeated three times. PTEN enzymatic assays utilized technical duplicates and was repeated using three HUVEC donors. All western blot and co-immunoprecipitation assays were conducted three to four times. Representative data shown