Fig. 2: ZFYVE21 sequesters Rubicon and RNF34 on Rab5 endosomes to enhance their stability.

HUVECs stably transduced with Rab5 WT or Rab5 DN were treated with PRA for the indicated times prior to I.F. analyses (a), co-IPs (b) and Western blotting (c). HUVECs were co-transduced with ZFYVE21-RFP, Rubicon-BNP, and RNF34-GFP constructs prior to I.F. analysis (d). HUVECs co-transduced with Rubicon and Rab5 or RNF34 and Rab5 constructs were analyzed following control or ZFYVE21 siRNA transfection (e). HUVECs were transfected with ZFYVE21 siRNA (f) or pre-treated vehicle or PIKIII (5 nM) prior to PRA treatment for 30 min prior to Western blot analysis (g). HUVECs were exposed to PRA in the presence or absence of cycloheximide (CHX, h, 10 μg/mL), MG132 (i, 25 μM), or leupeptin (j, 50 μM) for the indicated times prior to analysis by Western blotting. For Fig. 2h, p values involve comparisons of the respective timepoint between treatment with PRA and PRA plus cycloheximide where * indicates p < 0.05. For Fig. 2i, j, p-values involve comparisons of respective timepoint to timepoint zero where * indicates p < 0.05. HUVECs were transduced with Rab5 DN, pre-treated with MG132 and analyzed at the times indicated (k). Two-tailed Student’s t test (a, e) and one-way ANOVA (Fig. 2h-j) followed by Tukey’s pairwise comparison were used for statistical comparisons where p < 0.05 is considered statistically significant. Experiments repeated 3 times. All scale bars, 200 μm. Experiments repeated 3 times (a–k). Data are presented as mean values+/-SD. p-values are indicated in the figures.