Journal: Nature Communications

Article Title: A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells

doi: 10.1038/s41467-023-38684-2

Figure Lengend Snippet: IFN-γ-pretreated HUVECs were exposed to PRA at the indicated times prior to performing Western blotting ( a , g , j ) and Rab5 pulldowns ( b , e , h ). HUVEC stably transduced with Rab5 WT-GFP were treated with PRA for 30 min and subjected to proximity ligation assays (PLAs, c ). HUVECs were co-transduced with Rubicon-GFP or RNF34-GFP along with Rab5-RFP constructs, treated with PRA for 30 min and analyzed by confocal I.F. ( d , f , i ). Two-tailed Student’s t -test ( c , f , i ) was used for statistical comparisons where p < 0.05 is considered statistically significant. Experiments repeated 2 times ( c , f , i ), 3 times ( b , d , e , g , h , j ) , and 5 times ( a ) with different HUVEC donors. All scale bars, 200 μm. p -values are indicated in the figures.

Article Snippet: For protein co-incubation studies, Rubcion-GST (1 μg, Origene, #TP320593) and RNF34-His proteins (1 μg, Novus, #NBP2-23440) were co-incubated at 1:1 ratio at a volume of 50 μL at room temperature overnight with gentle rocking prior to resolution by SDS-PAGE.

Techniques: Western Blot, Stable Transfection, Transduction, Ligation, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells

doi: 10.1038/s41467-023-38684-2

Figure Lengend Snippet: 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.

Article Snippet: For protein co-incubation studies, Rubcion-GST (1 μg, Origene, #TP320593) and RNF34-His proteins (1 μg, Novus, #NBP2-23440) were co-incubated at 1:1 ratio at a volume of 50 μL at room temperature overnight with gentle rocking prior to resolution by SDS-PAGE.

Techniques: Stable Transfection, Transduction, Western Blot, Construct, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells

doi: 10.1038/s41467-023-38684-2

Figure Lengend Snippet: PRA-treated HUVECs were co-transduced with Rubicon-GFP and RNF34-RFP constructs and analyzed by confocal I.F. a HUVECs stably transduced with Rubicon-GFP were transiently transfected with increasing concentrations of RNF34-His plasmid prior to GFP co-IPs and Western blotting ( b ). HUVECs stably transduced with ZFYVE21-GFP were treated with PRA for 30 min ( c ). Rubicon (1μg) and RNF34 (1 μg) were co-incubated at 1 h at room temperature (lanes 1-3) or overnight at 4 °C (lanes 4-6) prior to resolution via SDS-PAGE ( d ). Purified ZFYVE21-GFP (1 μg) was used as a probe using recombinant human Rubicon (1 μg), RNF34 (1 μg), or a 1:1 mixture of Rubicon and RNF34 as bait ( e ). Rubicon-GST (1 μg), RNF34-His (1 μg), or a 1:1 mixture of Rubicon and RNF34 pre-incubated for 4 h at room temperature were added to ZFYVE21-GFP-conjugated agarose beads. Protein complexes were eluted and analyzed via Western blotting under non-denaturing conditions ( f ). Plate-bound ZFYVE21, Rubicon, or RNF34 were incubated with varying concentrations of prey proteins as indicated in ELISAs ( g ). GFP-tagged constructs were overexpressed in HeLa cells following by GFP co-IPs ( h , i ). Rab5 DN-mCherry was stably co-transduced with ZFYVE21-GFP ( j ) or Rubicon-GFP ( k ) in HUVECs and treated with MG132 as indicated prior to GFP co-IPs. p values involve relative comparisons with no prey protein added using one-way ANOVA followed by Tukey’s pairwise comparison where * indicates p < 0.05. Experiments repeated 2 times ( a , b , d – f , j , k ), 3 times ( c , j , k ) or 4 times ( g – i ) with different HUVEC donors. All scale bars, 200 μm. Data are presented as mean values+/-SD. p -values are indicated in the figures.

Article Snippet: For protein co-incubation studies, Rubcion-GST (1 μg, Origene, #TP320593) and RNF34-His proteins (1 μg, Novus, #NBP2-23440) were co-incubated at 1:1 ratio at a volume of 50 μL at room temperature overnight with gentle rocking prior to resolution by SDS-PAGE.

Techniques: Transduction, Construct, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Incubation, SDS Page, Purification, Recombinant

Journal: Nature Communications

Article Title: A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells

doi: 10.1038/s41467-023-38684-2

Figure Lengend Snippet: 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.

Article Snippet: For protein co-incubation studies, Rubcion-GST (1 μg, Origene, #TP320593) and RNF34-His proteins (1 μg, Novus, #NBP2-23440) were co-incubated at 1:1 ratio at a volume of 50 μL at room temperature overnight with gentle rocking prior to resolution by SDS-PAGE.

Techniques: Transfection, Over Expression, Plasmid Preparation, Stable Transfection, Transduction, Recombinant, Incubation, Western Blot

Journal: Nature Communications

Article Title: A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells

doi: 10.1038/s41467-023-38684-2

Figure Lengend Snippet: Human artery xenografts implanted in immunodeficient SCID/beige hosts were exposed to PRA for 24 h (200 μL i.v. tail vein injection per mouse) and analyzed in proximity ligation assays ( a , n = 3 per group). Sera from SCID/beige hosts bearing PRA-treated human artery xenografts were analyzed by Western blot ( b , n = 3 per group). HUVECs stably transduced with Rab5 WT mCherry or Rab5 DN mCherry ( c ), Rubicon shRNA ( d ) or RNF34 shRNA ( e ) were embedded in collagen gel matrices, implanted in SCID/beige mice for 2–4 weeks, and microvessels were exposed to PRA for 24 h (200 μL i.v. tail vein injection per mouse) prior to staining and analysis by I.F ( n = 3 per group). Sera from SCID/beige hosts implanted with collagen gel matrices were analyzed by Western blot ( f , n = 3 per group). C57/Bl6 or Rubicon-/-mice were injected with 750 μg MOPC or anti-H-2 b Abs ( g ), and kidneys were harvested for Rab5 co-IPs 24 h later ( n = 3 per group, h ). Figure 7g was created with BioRender.com. Two-tailed Student’s t -test was used for statistical comparisons ( a , c – e ) where p < 0.05 is considered statistically significant. Experiments repeated 3 times. All scale bars, 400 μm. p -values are indicated in the figures.

Article Snippet: For protein co-incubation studies, Rubcion-GST (1 μg, Origene, #TP320593) and RNF34-His proteins (1 μg, Novus, #NBP2-23440) were co-incubated at 1:1 ratio at a volume of 50 μL at room temperature overnight with gentle rocking prior to resolution by SDS-PAGE.

Techniques: Injection, Ligation, Western Blot, Stable Transfection, Transduction, shRNA, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Rubicon modulates neuroimmune responses following traumatic brain injury

doi: 10.64898/2026.03.04.709622

Figure Lengend Snippet: A Immunoblot comparing lysates of ipsilateral cortices from wild-type and Rubcn-mutant mice probed with Rubicon A (RbA) antibody. B Western blot showing immunoprecipitation of Rubicon protein from brain lysates of wild-type and Rubcn-mutant mice using Rubicon A (RbA) and Rubicon B (RbA) antibodies. * represents background signal. Blot is probed with RbB antibody. C Rubicon protein (amino-acid) sequence highlighting peptides (p1-p12 in blue) used for peptide mapping of wild-type and mutant RUBCN proteins. Red circle is highlighting Met296 as a potential alternative translation initiation site. D Normalized peptide abundance across the length of RUBCN protein in wild-type and Rubcn-mutant immunoprecipitates using RbA and RbB antibodies. E Comparative enrichment analysis showing fold change of proteins enriched in wild-type over Rubcn-mutant immunoprecipitates using RbA and RbB antibodies. F Immunoprecipitation assay showing lysates of HEK293T cells transfected with indicated plasmids for 24 h and immunoprecipitated with anti-FLAG or anti-HA agarose beads. Left: Immunoblot of immunoprecipitates with the indicated antibodies. Right: Fold change of NRROS signal in EGFP-Rubicon-Flag and Nrros-Myc/His (Nrros+Rubicon1-3) co-transfected cells to EGFP-C1 and Nrros-Myc/His (Nrros ONLY) co-transfected cells. Each FLAG IP sample was normalized to corresponding HA IP sample for quantification. Sample size (n) = 3 biological replicates for EGFP-Rubicon-Flag and Nrros-Myc/His co-transfected cells. G Immunoblot of whole cell lysates used for immunoprecipitation assay in F. H Immunoprecipitation assay showing lysates of HEK293T cells transfected with indicated plasmids for 24 h and immunoprecipitated with anti-MYC or anti-IgG antibodies. Left: Immunoblot of immunoprecipitates with the indicated antibodies. Right: Fold change of RUBCN signal in EGFP-Rubicon-Flag and Nrros-Myc/His (Nrros+Rubicon1-2) co-transfected cells to EGFP-C1 and EGFP-Rubicon-Flag (Rubicon ONLY) co-transfected cells. Each MYC IP sample was normalized to corresponding IgG IP sample. Sample size (n) = 2 biological replicates for EGFP-Rubicon-Flag and Nrros-Myc/His co-transfected cells. I Immunoblot of whole cell lysates used for immunoprecipitation assay in H.

Article Snippet: HEK293T cells were transiently transfected with EGFP-Rubicon (Addgene; 28022), EGFP-Rubicon Δ182 (Addgene; 28041), EGFP-Rubicon ΔCT (Addgene; 28040), pcDNA3.1(+)-human LRRC33 (Addgene; 111602), and/or pEGFP-C1 vectors using Lipofectamine TM 3000 Transfection Reagent (ThermoFisher Scientific; L3000015) for 24 h.( , , ) The cell monolayers were washed with ice-cold PBS and lysed in cell lysis bucer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 or 0.5% Igepal CA-630 (SigmaAldrich; I8896) containing protease and phosphatase inhibitors.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Sequencing, Transfection

Journal: bioRxiv

Article Title: Rubicon modulates neuroimmune responses following traumatic brain injury

doi: 10.64898/2026.03.04.709622

Figure Lengend Snippet: A Immunoblot comparing lysates of ipsilateral cortices from wild-type and Rubcn-mutant mice probed with Rubicon A (RbA) antibody. B Western blot showing immunoprecipitation of Rubicon protein from brain lysates of wild-type and Rubcn-mutant mice using Rubicon A (RbA) and Rubicon B (RbA) antibodies. * represents background signal. Blot is probed with RbB antibody. C Rubicon protein (amino-acid) sequence highlighting peptides (p1-p12 in blue) used for peptide mapping of wild-type and mutant RUBCN proteins. Red circle is highlighting Met296 as a potential alternative translation initiation site. D Normalized peptide abundance across the length of RUBCN protein in wild-type and Rubcn-mutant immunoprecipitates using RbA and RbB antibodies. E Comparative enrichment analysis showing fold change of proteins enriched in wild-type over Rubcn-mutant immunoprecipitates using RbA and RbB antibodies. F Immunoprecipitation assay showing lysates of HEK293T cells transfected with indicated plasmids for 24 h and immunoprecipitated with anti-FLAG or anti-HA agarose beads. Left: Immunoblot of immunoprecipitates with the indicated antibodies. Right: Fold change of NRROS signal in EGFP-Rubicon-Flag and Nrros-Myc/His (Nrros+Rubicon1-3) co-transfected cells to EGFP-C1 and Nrros-Myc/His (Nrros ONLY) co-transfected cells. Each FLAG IP sample was normalized to corresponding HA IP sample for quantification. Sample size (n) = 3 biological replicates for EGFP-Rubicon-Flag and Nrros-Myc/His co-transfected cells. G Immunoblot of whole cell lysates used for immunoprecipitation assay in F. H Immunoprecipitation assay showing lysates of HEK293T cells transfected with indicated plasmids for 24 h and immunoprecipitated with anti-MYC or anti-IgG antibodies. Left: Immunoblot of immunoprecipitates with the indicated antibodies. Right: Fold change of RUBCN signal in EGFP-Rubicon-Flag and Nrros-Myc/His (Nrros+Rubicon1-2) co-transfected cells to EGFP-C1 and EGFP-Rubicon-Flag (Rubicon ONLY) co-transfected cells. Each MYC IP sample was normalized to corresponding IgG IP sample. Sample size (n) = 2 biological replicates for EGFP-Rubicon-Flag and Nrros-Myc/His co-transfected cells. I Immunoblot of whole cell lysates used for immunoprecipitation assay in H.

Article Snippet: HEK293T cells were transiently transfected with EGFP-Rubicon (Addgene; 28022), EGFP-Rubicon Δ182 (Addgene; 28041), EGFP-Rubicon ΔCT (Addgene; 28040), pcDNA3.1(+)-human LRRC33 (Addgene; 111602), and/or pEGFP-C1 vectors using Lipofectamine TM 3000 Transfection Reagent (ThermoFisher Scientific; L3000015) for 24 h.( , , ) The cell monolayers were washed with ice-cold PBS and lysed in cell lysis bucer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 or 0.5% Igepal CA-630 (SigmaAldrich; I8896) containing protease and phosphatase inhibitors.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Sequencing, Transfection