excess sulfo succinimidyl 4 formylbenzoate s 4fb  (Vector Laboratories)

Journal: The Journal of Biological Chemistry

Article Title: Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets

doi: 10.1016/j.jbc.2026.111191

Figure Lengend Snippet: 19-Atriol inhibits progesterone biosynthesis independently of TSPO and acts downstream of cholesterol import. A , schematic of the initial steps in the steroidogenic pathway: (i) Mitochondrial cholesterol import is mediated by the intermembrane space shuttle STAR. (ii) Cholesterol is converted to pregnenolone/P5 by CYP11A1 in the mitochondrial matrix. (iii) P5 is further converted to progesterone/P4 by 3β-hydroxysteroid dehydrogenase (HSD3B) at the endoplasmic reticulum. B , dose–response analysis of progesterone production in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells stimulated with Bt 2 cAMP. Increasing concentrations of 19-Atriol reduced P4 output in both genotypes (∗ p < 0.05) with comparable potency and maximal inhibition, indicating that its pharmacological action is independent of TSPO. C , immunoblot analysis of STAR and CYP11A1 expression following treatment with 19-Atriol (0,10 and 100 μM). TSPO was absent in MA-10: Tspo Δ/Δ cells, validating the knockout. Neither STAR nor CYP11A1 protein abundance was altered by 19-Atriol in either genotype (ACTB served as a loading control). D , Dose-response analysis of progesterone production in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells with supplementation of 22R-HC in the absence of stimulation. 19-Atriol continued to inhibit P4 production in both genotypes (∗ p < 0.05), indicating that the observed inhibitory effect occurs downstream of cholesterol import.

Article Snippet: Briefly, supernatants were incubated overnight at 4 °C with 125 I-labeled progesterone (MP Biomedicals) and an anti-progesterone antibody ( ) for competitive binding.

Techniques: Inhibition, Western Blot, Expressing, Knock-Out, Quantitative Proteomics, Control

Journal: The Journal of Biological Chemistry

Article Title: Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets

doi: 10.1016/j.jbc.2026.111191

Figure Lengend Snippet: 19-Atriol is metabolized by HSD3B to 19-OHT, and 19-OHT also inhibits progesterone synthesis. A , reaction scheme showing 19-Atriol is converted by 3β-hydroxysteroid dehydrogenase (HSD3B) to 19-hydroxytestosterone (19-OHT). LC–MS/MS showed dose-dependent accumulation of 19-OHT with increasing 19-Atriol (1–100 μM) in MA-10 cells under basal conditions and with Bt 2 cAMP stimulation, indicating that 19-Atriol serves as an HSD3B substrate. B , similar to 19-Atriol, 19-OHT decreased P4 output in a dose-dependent manner in wild-type MA-10 and TSPO-null MA-10: Tspo Δ/Δ cells with comparable potency and maximal effect, with significant differences between the indicated concentrations (∗ p < 0.05). C , with 22R-HC supplementation, 19-OHT still reduced P4 production in both genotypes in a dose-dependent fashion similar to 19-Atriol, with significant differences between the indicated concentrations (∗ p < 0.05). D , in P5-supplemented assays, 19-OHT (0, 10, 100 μM) suppressed P4 formation at each P5 input (1, 5, and 10 μM). Distinct letters above bars indicate statistically significant differences within each P5 condition ( p < 0.05).

Article Snippet: Briefly, supernatants were incubated overnight at 4 °C with 125 I-labeled progesterone (MP Biomedicals) and an anti-progesterone antibody ( ) for competitive binding.

Techniques: Liquid Chromatography with Mass Spectroscopy