β tubulin  (Bio-Rad)

Journal: Frontiers in Pharmacology

Article Title: Soluble epoxide hydrolase inhibition restores pro-resolving lipid mediators and reduces inflammation in localized provoked vulvodynia

doi: 10.3389/fphar.2026.1741914

Figure Lengend Snippet: sEH is involved in the LPV inflammatory process. (A) Illustration of punch biopsy collection from LPV cases and healthy controls during surgery. Light touch to the vulvar vestibule (Vest) is immensely painful, whereas the adjacent external vulva (Vulv) is not painful to touch. (B) RT-qPCR data showing sEH (EPHX2) mRNA expression, normalized to 18S rRNA, in LPV and control fibroblasts (n = 6 cases and 6 controls). (C) Western blot data showing sEH (EPHX2) protein expression, normalized to β-tubulin, in LPV and control fibroblasts (n = 3 cases and 3 controls). (D) sEH enzyme activity is significantly elevated in the LPV vest compared to LPV vulv, control vest, and control vulv (n = 3 cases and 3 controls). The data was collected in technical triplicates and are represented as mean ± SEM, one-way ANOVA + Tukey’s post hoc test, *P < 0.05, ns = not significant. (E) siRNA knockdown with sEH siRNA [5 µg] in vulvar and vestibular fibroblasts derived from 3 LPV cases was confirmed through Western blotting. (F) Transfection of sEH siRNA in LPV case vestibular and vulvar fibroblasts, but not control siRNA, reduced IL-6 and PGE2 production upon IL-1β [500 pg/mL] stimulation (n = 3 cases). The data was collected in six technical replicates and are represented as mean ± SEM, two-way ANOVA + Tukey’s post hoc test, *P < 0.05.

Article Snippet: Protein blots were probed with EPHX2 rabbit polyclonal antibody (Abclonal, Woburn, MA, Cat: A1885) at a dilution of 1:1,000, hFAB Rhodamine Anti-Tubulin Primary Antibody (Bio-Rad, Cat: #12004166) at a dilution of 1:5,000, and peroxidase AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, Cat: 111-035-003) at a dilution of 1:4,000.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Activity Assay, Knockdown, Derivative Assay, Transfection

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