hfabtm rhodamine anti gapdh primary antibody  (Bio-Rad)

Journal: Frontiers in Immunology

Article Title: Upregulation of miR-146a-5p and miR-146b-5p limits IL-1β-mediated signaling in adipose tissue during polytrauma by downregulating IRAK1

doi: 10.3389/fimmu.2026.1658504

Figure Lengend Snippet: MiR-146a-5p suppresses IL-1β-induced proinflammatory signaling by targeting IRAK1 (A) Venn diagram representing the intersection of two in silico target gene predictions (TargetScan 8.0 and miRWalk 3.0) for miR-146a-5p and genes of the IL-1β signaling pathway (WikiPathway 195) resulting in an overlap of three genes, i.e. IRAK1, TRAF6, REL. (B, C) SGBS adipocytes were transfected with 50 nM miR-146a-5p mimic or a non-targeting control (NC). (B) Total RNA was isolated 7 days post-transfection. IRAK1 , TRAF6 , and REL mRNA levels were assessed by RT-qPCR using the ΔCt-method with HPRT as reference gene. The results are displayed as mean ± SEM of four independent experiments performed in triplicates. (C) Protein was extracted 7 days post-transfection. Protein expression of IRAK1, TRAF6 and c-REL was assessed by Western Blot with GAPDH as corresponding loading control. One representative Western Blot out of four independent experiments is shown. Densitometric analyses is displayed as mean ± SEM of four independent experiments performed in duplicates. (D) HEK293 cells were transfected as indicated (plasmid amount used: 25 ng, miRNA amount used: 100 nM). Next, a dual luciferase reporter gene assay was performed by determining the luciferase signal expressed as Firefly over Renilla, denoted in (a.u.). The results are displayed as mean ± SEM of three independent experiments performed in triplicates. (E) SGBS adipocytes were transfected with 20 nM IRAK1 siRNA or control (Ctrl). mRNA levels were assessed by RT-qPCR using the ΔCt-method with HPRT as reference gene 72 h post-transfection. The results are displayed as mean ± SEM of four independent experiments performed in triplicates. (F) siRNA-transfected SGBS adipocytes were stimulated with IL-1β (200 pg/ml) or the corresponding vehicle control 72 h post-transfection. Total RNA was isolated 4 h after stimulation. IL6, IL8, and MCP1 mRNA expression was analyzed. Statistics: paired two-tailed t-test (B, C, E) , one-way ANOVA with Tukey correction, *p < 0.05, **p < 0.01 (D) , two-way ANOVA with Šídák correction, *p < 0.05 (F) .

Article Snippet: Adiponectin (GTX112777, polyclonal, rabbit, GeneTex, dilution 1:1,000), c-REL (82000, monoclonal, rabbit, Cell Signaling Technology (CST), dilution 1:1,000), IRAK1 (SC-5287, monoclonal, mouse, Santa Cruz Biotechnology, dilution 1:200), pAKT (S473) (9271, polyclonal, rabbit, CST, dilution 1:1,000), AKT (9272, polyclonal, rabbit, CST, dilution 1:1,000), pERK 1/2 (Thr202/Tyr204) (9106, monoclonal, mouse, CST, dilution 1:2,000), ERK 1/2 (M5670, polyclonal, rabbit, Sigma-Aldrich, dilution 1:10,000), GLUT4 (PA1722, polyclonal, rabbit, Boster, dilution 1:1,000), pIκBα (Ser32/36) (9246, monoclonal, mouse, CST, dilution 1:1,000), IκBα (Ser32/36) (9242, polyclonal, rabbit, CST, dilution 1:1,000), Leptin (RD181001220, polyclonal, rabbit, bioVendor, dilution 1:1,000), PLIN1 (Ab3526, polyclonal, rabbit, Abcam, dilution 1:1,000), PPARγ (2443, monoclonal, rabbit, CST, dilution 1:1,000), TRAF6 (PA5-29622, polyclonal, rabbit, Invitrogen, dilution 1:1,000) and GAPDH (12004168, Rhodamine, Bio-Rad, dilution 1:5,000) were used as primary antibodies.

Techniques: In Silico, Transfection, Control, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Luciferase, Reporter Gene Assay, Two Tailed Test