plasmid pcdna3 1 egfp  (Addgene inc)

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