Journal: bioRxiv

Article Title: Rubicon modulates neuroimmune responses following traumatic brain injury

doi: 10.64898/2026.03.04.709622

Figure Lengend Snippet: A Variant sparse Partial Least Squares-Discriminate Analysis (sPLS-DA) plot demonstrating group separation among ipsilateral cortical samples from wild-type sham, Rubcn-mutant sham, wild-type injured, and Rubcn-mutant injured mouse groups. B Volcano plots highlighting differentially expressed genes (DEGs) between injured and sham mice in wild-type (left) and Rubcn-mutant (right) samples, with red representing significantly upregulated genes and blue representing significantly downregulated genes. C Volcano plots showing DEGs between Rubcn-mutant and wild-type mice in sham (left) and injured samples (right). A data point corresponding to the Rubcn gene has been removed from the right volcano plot to minimize the y-axis. D Heatmap of gene expression across the samples demonstrating top upregulated (top-half) and downregulated (bottom-half) DEGs between Rubcn-mutant and wild-type TBI mice. E Gene Ontology Biological Process (GO:BP) analysis of significant DEGs (P-value<0.01) between Rubcn-mutant and wild-type TBI samples. Top graph shows GO-terms associated with upregulated genes (in orange) and bottom graph shows GO-terms associated with downregulated genes (in blue) in Rubcn-mutant over wild-type TBI samples with FDR<0.05. F Heat map of selected TBI-induced inflammatory DEG expression across wild-type and Rubcn-mutant sham and TBI samples. G Ingenuity Pathway Analysis (IPA) analysis comparing canonical pathways activated following TBI in wild-type versus Rubcn-mutant mice. Orange (activated) and blue (inhibited) indicate significantly enriched canonical pathways based on activation z-score, while gray dots represent pathways detected in the specified dataset that did not meet significance thresholds. H IPA analysis of lipid-related pathways activated following TBI in wild-type versus Rubcn-mutant mice. Sample size (n) were as follows: WT Sham=5, WT TBI D3=5, Rubcn Mut. Sham=4, Rubcn Mut. TBI D3=6.

Article Snippet: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

Techniques: Variant Assay, Mutagenesis, Gene Expression, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Rubicon modulates neuroimmune responses following traumatic brain injury

doi: 10.64898/2026.03.04.709622

Figure Lengend Snippet: A Heatmap of RT-qPCR data showing expression of inflammatory genes in ipsilateral cortices of wild-type and Rubcn -mutant mice at 1 dpi (TBI D1) and 3 dpi (TBI D3) in comparison to sham. Color coding is based on z-score. B Fold change in expression levels of indicated genes in wild-type and Rubcn -mutant mice corresponding to A . C Heatmap of RT-qPCR data showing expression of inflammatory genes in ipsilateral cortices of wild-type and Rubcn -mutant mice at 29 dpi (TBI D29). D Fold change in expression levels of indicated genes in wild-type and Rubcn -mutant mice corresponding to C. Bars represent mean ± s.e.m. Sample size ( n ) for A-B were as follows: WT Sham=8, WT TBI D1=5, WT TBI D3=9, Rubcn Mut. Sham=8, Rubcn Mut. D1=7, Rubcn Mut. D3=9. Sample size ( n ) for C-D were as follows: WT Sham=6, WT TBI D29=6, Rubcn Mut. Sham=7, Rubcn Mut. TBI D29=5. 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: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Rubicon modulates neuroimmune responses following traumatic brain injury

doi: 10.64898/2026.03.04.709622

Figure Lengend Snippet: A Number of foot faults before and after injury in wild-type and Rubcn-mutant mice by beam walk test. B Percent of one/two-foot support (left) and three/four-foot support (right) after injury in wild-type and Rubcn-mutant mice by assessed by CatWalk test. C Percentage of ipsilateral cortical damage at 29 dpi between injured wild-type and Rubcn-mutant mice. Data represent mean ± s.e.m. Sample size (n) for A were as follows: WT Sham=11, WT TBI=13, Rubcn Mut. Sham=13, Rubcn Mut. TBI=14. Sample size (n) for B were as follows: n=7-14 mice per group as indicated. Sample size (n) for C were as follows: WT TBI=5, Rubcn Mut. TBI=5. Statistical Analyses: For A-B, mixed-effects analysis with post hoc Tukey’s test; For C, Unpaired Student’s t-test. For all data, significance is represented by * P-value<0.05, ** P-value<0.005.

Article Snippet: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

Techniques: Mutagenesis

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: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

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

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: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

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 of ipsilateral cortical tissue lysates from wild-type and Rubcn-mutant mice comparing lipid peroxidation marker at 1 dpi. B Densitometric analysis of immunoblot in A. C Immunoblot of ipsilateral cortical tissue lysates from wild-type and Rubcn-mutant mice comparing lipid peroxidation marker at 3 dpi. D Densitometric analysis of immunoblot in C. Data represent mean ± s.e.m. Sample size (n) were as follows: WT Sham=6, WT TBI D1=6, WT TBI D3=6, Rubcn Mut. Sham=6, Rubcn Mut. D1=6, Rubcn Mut. D3=6.Statistical analyses: 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: The following antibodies were used for immunobloting: RUBCN (CST-8465S; 68261S), NRROS (CST; 34388S), NLRP3 (CST; 15101S), cGAS (CST; 15102S), STING (CST; 13647S), α-fodrin (BML-FG6090-0100), P62 (BD Biosciences; 610832), LC3(CST; 2775S; NB100-2220), GAPDH (CST; 2118S), HMGB1(CST; 3935S), FLAG (CST; 33542S), MYC (CST; 2276S), HIS (CST; 2365S), GFP (CST; 2955S), 4-HNE (R&D Systems; MAB3249), mouse IgG1(CST; 5415S), mouse IgG2a (CST; 61656), anti-rabbit IgG-HRP (CST; 7074S), and anti-mouse IgG-HRP (CST; 7076S) Uncropped immunoblots are presented if Fig. S5 .

Techniques: Western Blot, Mutagenesis, Marker

Journal: PLOS Biology

Article Title: CUL5 E3 ubiquitin ligase regulates the evasion of bladder cancer cells to CD8 + T cell-mediated killing by inhibiting autophagy

doi: 10.1371/journal.pbio.3003647

Figure Lengend Snippet: (A) The expression levels of LC3B and P62 in T24 cells transfected with vector, Flag-Rubicon-L, or Flag-Rubicon-S were detected by western blotting. (B) T24 cells were transfected with vector, Flag-Rubicon-L or Flag-Rubicon-S plasmids, and those co-transfected with mCherry-EGFP-LC3B. The autophagosomes with yellow puncta and autolysosomes with red puncta. Bar: 10 μm. (C) T24 cells were transfected with the vector, Flag-Rubicon-L, and Flag-Rubicon-S plasmids, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (D) Western blotting with the indicated antibodies in T24 cells transfected with scramble, sh-RUBCN-L or sh-RUBCN-S. The efficiency of RUBCN-L knockdown and RUBCN-S knockdown in T24 cells was detected by agarose gel electrophoresis (left) and qRT-PCR (right). (E) Co-IP assay using antibody specific for Rubicon showed that Rubicon interacted with UVRAG and Beclin1 in T24 cells. The precipitate was subjected to western blotting with the antibodies against Rubicon, UVRAG, and Beclin1. (F) Co-IP assay using antibody specific for Flag showed that Flag-Rubicon-S interacted with UVRAG and Beclin1(Right), while Flag-Rubicon-L could not bind to UVRAG and Beclin1 in T24 cells (Left). The precipitate was subjected to western blotting with the antibodies against Flag, UVRAG, and Beclin1. (G) The expression levels of LC3B and P62 in CUL5-KO T24 cells were detected by western blotting. (H) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the mCherry-EGFP-LC3B. The autophagosomes with yellow puncta and autolysosomes with red puncta. Bar: 10 μm. (I) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-PTBP1#1. (J) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-RUBCN-L, and agarose gel electrophoresis for analysis of RUBCN isoforms. (K) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the scramble or sh-RUBCN-L, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (L) Western blotting with the indicated antibodies in CUL5-WT and CUL5-KO T24 cells transfected with scramble or sh-RUBCN-S, and agarose gel electrophoresis for analysis of RUBCN isoforms. (M) CUL5-WT, CUL5-KO#1, and CUL5-KO#2 T24 cells were transfected with the scramble or sh-RUBCN-S, and then co-cultured with CD8 + T cells, and cell viability was measured by CCK-8. (N) The expression levels of MHC-I (HLA-A, -B, -C) in CUL5-KO T24 cells co-cultured with CD8 + T cells were detected by western blotting. Data are presented as the means ± SD from three independent experiments. Student t test was applied to analyze and compare the data in C, D, K, and M. ns, nonsignificant; ** P < 0.01; *** P < 0.001. The raw data underlying all figures can be found in . Original blots and gels can be found in .

Article Snippet: Antibodies used included primary antibodies against CUL5 (Abclonal, A5369), β-Actin (Proteintech, 66009-1-Ig), PRMT5 (Proteintech, 18436-1-AP), THOC2 (Proteintech, 55178-1-AP), THRAP3 (Proteintech, 19744-1-AP), SNRNP200 (Proteintech, 23875-1-AP), PTBP1 (Proteintech, 12582-1-AP), SF3B1(Proteintech, 27684-1-AP), PRPF8(Proteintech, 11171-1-AP), SF3B2 (Proteintech, 10919-1-AP), HNRNPC (Proteintech, 11760-1-AP), LC3B (Abclonal, A5618), P62 (Proteintech, 18420-1-AP), Rubicon (Proteintech, 21444-1-AP), UVRAG (Proteintech, 29190-1-AP), Beclin1 (Proteintech, 11306-1-AP), HLA-class I (Proteintech, 15240-1-AP), Rabbit control IgG (Abclonal, AC005), Mouse control IgG (Abclonal, AC011), Mouse anti-HA tag (Abclonal, AE008), Rabbit anti-HA tag (Abclonal, AE036), Mouse anti-Flag tag (Abclonal, AE005), and Rabbit anti-Flag tag (Abclonal, AE004); HRP-conjugated secondary goat anti-mouse (Proteintech, SA00001-1), or goat anti-rabbit (Proteintech, SA00001-2) antibodies.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Culture, CCK-8 Assay, Knockdown, Agarose Gel Electrophoresis, Quantitative RT-PCR, Co-Immunoprecipitation Assay