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
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