anti pgsk3β tyr216  (Proteintech)

Journal: Redox Biology

Article Title: Persulfidation alters gene regulatory programs and promotes endothelial specification

doi: 10.1016/j.redox.2025.103926

Figure Lengend Snippet: Sulfide supplementation during mesodermal commitment promotes vascular development through activating WNT signaling, without impacting on redox balance. (A) Normalized cell number in cell cycle phases detected by live cell imaging of FUCCI lentivirally induced mesodermal cells following treatment with 0–10 μmol/L Na 2 S 3 for up to 48 h. (B) MitoSOX levels in mesodermal cells treated with solvent (Sol.) or with 5 μmol/L Na 2 S 3 for 48 h. (C) Immunoblotting of 3-nitrotyrosine (3-NT) and vinculin (VIN) in mesodermal cells treated with solvent (Sol.) or with 5 μmol/L Na 2 S 3 for 48 h. (D) Volcano plot showing persulfidated proteins in mesodermal cells treated with solvent (Sol.) or with 5 μmol/L Na 2 S 3 for 48 h. (E) Enrichment analysis of proteins detected as in panel D. (F) Heatmap showing transcriptional changes following RNA sequencing in samples as in panel D. (G) Gene set enrichment analysis following RNA sequencing in samples as in panel D. (H) β-catenin stability and phosphorylation of GSK3β at Ser9 in mesodermal cells treated with solvent (Sol.) or with 5 μmol/L Na 2 S 3 for 24 h and representative immunoblots at 24 h. (I) Transcription factor (TF) activities following RNA sequencing in samples as in panel D. (J) Representative immunofluorescent image of human iPSC-derived vascular organoids receiving solvent (Sol.) or 5 μmol/L Na 2 S 3 after mesodermal specification. bar = 200μm. 3 iPSC lines were used in 2–3 technical replicates. Graphs include the average of all experiments per line. Paired Student's t-test (B, C, D), Two-way ANOVA, Tukey's multiple comparison test (A, H).

Article Snippet: The following antibodies were used: mouse anti-3-nitrotyrosine (3-NT) mAb (1:1000, #ab110282, Abcam), mouse anti-vinculin mAb (1:1000, ThermoScientific, #MA5-11690), rabbit anti-pGSK3β (Ser9) (1:1000, Cell Signaling Technology, Cat. #9336, monoclonal, human reactive), anti–β-catenin (1:1000, Cell Signaling Technology, Cat. #8480, monoclonal, human reactive), rabbit anti-SMAD2/3 (1:1000, Cell Signaling Technology, Cat. #8685, monoclonal, human reactive) andrabbit anti p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (1:1000, Cell Signaling Technology, Cat. #8828, monoclonal, human reactive).

Techniques: Live Cell Imaging, Solvent, Western Blot, RNA Sequencing, Phospho-proteomics, Derivative Assay, Comparison

Journal: Toxics

Article Title: Co-Exposure to Bisphenol A and a High-Fat Diet Induces Insulin Resistance via Suppression of Insulin Signaling Molecule Expression and GLUT4 Translocation

doi: 10.3390/toxics14020146

Figure Lengend Snippet: Effects of BPA and HFD on signaling molecule expression in gastrocnemius tissue in male and female mice. Results are presented as mean ± SD. Western blot bands of AKT, pAKT Ser473 , and GADPH protein level ( a ), with quantitative analysis of AKT ( n = 3) ( b ) and pAKT Ser473 ( n = 3) ( c ). ( d ) Western blot bands of Rab8A, VAMP2, Syntaxin4, GLUT4, and GADPH protein level, with quantitative analysis of Rab8A ( n = 3) ( e ), VAMP2 ( n = 3) ( f ), Syntaxin4 ( n = 3) ( g ), and GLUT4 ( n = 3) ( h ). Results of GLUT4 immunohistochemistry ( i ); quantitative analysis of immunohistochemistry results ( j ) ( n = 3). ( k ) Western blot bands of GSK3β, pGSK3β Ser9 , and GADPH protein levels, with quantitative analysis of GSK3β ( n = 3) ( l ) and pGSK3β Ser9 ( n = 3) ( m ). Compared with the C 0 group, * p < 0.05; compared with the C 1 group, # p < 0.05; compared with the C 2 group, △ p < 0.05.

Article Snippet: Proteins were incubated with monoclonal antibodies to AKT (CST, Beverly, MA, USA, 4685, 1:1000), p-AKT Ser473 (CST, Beverly, MA, USA, 4060, 1:1000), p-AKT Thr308 (CST, Beverly, MA, USA, 13038, 1:1000), Rab8A (Abcam, Cambridge, UK, ab188674, 1:1000), Rab13 (Merck, Darmstadt, Germany, SAB4200057, 1:1000), Syntaxin4 (Abcam, Cambridge, UK, ab184545, 1:1000), VAMP2 (Abcam, Cambridge, UK, ab181869, 1:5000), GLUT4 (Abcam, Cambridge, UK, ab33780, 1:1000), GSK3β (CST, Beverly, MA, USA, 12456, 1:1000), pGSK3β Ser9 (CST, Beverly, MA, USA, 9322, 1:1000), and GAPDH (Abcam, Cambridge, UK, ab8245, 1:1000),and subsequently incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibodies.

Techniques: Expressing, Western Blot, Immunohistochemistry