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

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 damage-associated marker (α-fodrin) and autophagy markers (P62, LC3II) in ipsilateral cortices from wild-type and Rubcn-mutant mice at 3 dpi to sham. B Densitometric quantification of immunoblot in A. C Immunoblot of ipsilateral hippocampus at 1 and 3 dpi in wild-type and Rubcn-mutant mice probed with autophagy markers. D Densitometric quantification of immunoblot in C. E Immunoblot of ipsilateral hippocampus at 1 dpi (TBI D1) and 3 dpi (TBI D3) in wild-type and Rubcn-mutant mice probed with damage-associated markers. F Densitometric quantification of immunoblot in E. G Immunofluorescence (IF) images of GFP-LC3 (green) coronal sections from wild-type and Rubcn-mutant sham and injured mice at 3 dpi stained for IBA1 (in yellow) and P62 (in magenta). Scale bar represents 50 μm. H Quantification of mean fluorescence intensity of P62 (left) and percent of P62/SQSTM1+ cells within LC3+/IBA+ cells (right) from G. All bars represent mean ± s.e.m. Sample size (n) for A-B were as follows: WT Sham=6, WT TBI D3=6, Rubcn Mut. Sham=6, Rubcn Mut. D3=6. Sample size (n) for C-F were as follows: WT Sham=6, WT TBI D1=6, WT TBI D3=6, Rubcn Mut. Sham=5, Rubcn Mut. D1=5, Rubcn Mut. D3=5. Sample size (n) for G-H were as follows: WT Sham=4, WT TBI D3=4, Rubcn Mut. Sham=4, Rubcn Mut. D3=4. Statistical analyses for all data: Two-way ANOVA with post hoc Tukey’s test and significance is represented by * P-value<0.05, ** P-value<0.005, *** P-value<0.0005, **** P-value<0.0001.

Article Snippet: GFP-LC3 (tg/+) Rubicon mutant mice were generated by crossing GFP-LC3 and Rubicon mutant mice at the UMB animal facility and were genotyped according to the Jackson Laboratory genotyping protocol.

Techniques: Western Blot, Marker, Mutagenesis, Immunofluorescence, Staining, Fluorescence