Fig. 5: Rubicon and RNF34 cooperatively reduce the stability of Rab5-associated fliI.

HUVECs were exposed to PRA for the indicated times prior to performing pulldowns for FliI (a) and caspase-1 (b). HeLa cells were transiently transfected with various GFP-tagged overexpression vectors prior to GFP co-IPs (c). HUVECs were transiently transfected with increasing plasmid concentrations encoding AA 625-760 of Rubicon fused with GFP, (d) or full-length FliI (e) followed by pulldowns for caspase-1. HUVECs stably transduced with Ubiquitin WT-HA (Ub-HA) were exposed to PRA for the indicated times followed by pulldowns for FliI (f). Ub-HA HUVECs were transfected with control or RNF34 siRNA as indicated and treated with PRA for 30 min (g). HUVECs were transfected with siRNA as indicated followed by Rab5 co-IPs following PRA treatment for 30 min (h). In ubiquitin ligase assays, recombinant human FliI (0.5 μg) was incubated as substrate with activated Ub, the indicated E2 ligase(s), and native RNF34 protein (0.5 μg, i) or RNF34 truncation mutants prior to Western blotting (j). Experiments repeated 3 times with different HUVEC donors.