Fig 1: NUMBL promotes C9 aggregates within Rab7+ endosomes.Rab5-GFP HUVECs were treated with PRA spiked with pHrodo-labeled C9 and analyzed by live cell imaging (a). HUVECs were treated with PRA for 1 h, washed, and analyzed 1 h afterwards by I.F. (b). PRA-treated HUVECs were sonicated, and vesicular fractions were enriched by sucrose gradient centrifugation, then analyzed by FACS at the times indicated (c). PRA-treated HUVECs were analyzed by I.F. (d) or by Western blot following Rab5 pulldowns at 1 h (e). HUVECs were transduced with Rab5 WT and Rab5 DN constructs, treated with PRA for 2 h prior to I.F., and punctae per cell were quantified (f). HUVECs were exposed to vehicle, bafilomycin (100 nM), or NH4Cl (1 μM) for 2 h, PRA was added, and thioflavin fluorescence was assessed 4 h later (g). Data points indicate technical replicates for cell regions (b) and cell punctae (d, f, g). Experiments repeated n = 2 (e), n = 3 (a, c, d, f, g), or n = 5 (b) times using different HUVEC donors. Error bars indicate standard deviations. Scale Bar = 30 μm (a, b), 20 μm (f, g). One-way ANOVA with Tukey’s post-hoc correction (a, b, g) or paired Student’s t-test (d, f).
Fig 2: NUMBL is a Rab35 effector promoting intracellular C9.HUVECs were treated with PRA for 1 h, and I.F. was performed with or without a Triton-X permeabilization prior to I.F. staining (a). HUVECs were treated with PRA (b) or MG132 (c) and analyzed by Western blot. LC-MS/MS analysis of ZFYVE21-GFP vesicles (d). PRA-treated HUVECs were analyzed by I.F. (e, f). NUMBL protein was tested for binding to C9 (g) and C9 mutant proteins (h, 1 μg/mL) by ELISA. HUVECs were treated with PRA for 1 h, and intracellular C9 was assessed by FACS with siRNA against Rab35 or NUMBL (i). Pulldowns of Rab35 (j) and Western blots of PRA-treated HUVECs (k). GTPγs or GDP was incubated with Rab35 for 30 min prior to addition of NUMBL for 30 min at RT prior to Western blot (l). Data points indicate technical replicates for number of cells analyzed (a, e) or number of sample wells (g–i). Error bars indicate standard deviations. Scale Bar = 30 μm (a, e–f). Experiments repeated n = 3 (b, c, j–l) times. Paired Student’s t-test (a, e) or one-way ANOVA with Tukey’s post-hoc correction (g–i).
Fig 3: NUMBL stabilizes ZFYVE21 to promote C9 aggrephagy.HUVECs were treated with PRA and analyzed by Western blot (a–c). HUVECs transduced with ZFYVE21-FLAG and LC3B-RFP were treated with PRA containing C9-AF647 protein (10 μg/mL) and either with or without CQ for 2 h prior to analysis by I.F. (d). HUVECs transduced with ZFYVE21-GFP were treated with PRA containing AF647-labeled C9 prior to live cell imaging (e). HUVECs were transfected with ZFYVE21 siRNA and analyzed in pulse-chase studies at various times after PRA treatment (f). HUVECs transduced with mRFP-GFP-LC3 constructs and transfected with ZFYVE21 siRNA were analyzed by I.F. after PRA treatment for the indicated times (g). HUVECs transduced with ZFYVE21 were treated with PRA and with or without CQ prior to ZFYVE21 co-IPs and Western blot analysis (h). HUVECs were transduced with FLAG-tagged ZFYVE21 constructs containing alanine substitutions at the amino acid sites indicated prior to FLAG co-IPs and Western blot analysis (i). Data points indicate technical replicates for cell punctae (d, g) or sample wells (i). Experiments were repeated n = 2 (b, c, h, i) or n = 3 (a, d, e, f) times using different HUVEC donors. Error bars indicate standard deviations. Scale Bar = 30 μm (d) or 40 μm (e). One-way ANOVA with Tukey’s post-hoc correction (a, d, e, f, h, i) or paired Student’s t-test (g).
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