foxo1/fkhr [p ser256] antibody  (Bio-Techne corporation)

Journal: Cell death discovery

Article Title: The inhibition of FKBP5 protects β-cell survival under inflammation stress via AKT/FOXO1 signaling.

doi: 10.1038/s41420-023-01506-x

Figure Lengend Snippet: Fig. 5 FOXO1 mediated the protective effect of FKBP5 inhibition on β cells. A, B Western blot analysis of Pdx1, p-Foxo1Ser256, Foxo1, p-Akt Ser473 and Akt in NIT-1 cells transfected with siFoxo1 or control siRNAs, and then treated with SAFit2 (A). Relative proteins expression levels were quantified by Image J (B). C, D Annexin-V/7-AAD staining and flow cytometry analysis in NIT-1 cells transfected with siFoxo1 or control siRNAs and then treated with cytokines, or cytokines + SAFit2 (C). Quantification of PE+7AAD+/PE+7AAD- cells (D). E, F Western blot analysis of FOXO1, BCL2, BAX in primary human islets transfected with siFOXO1 or control siRNAs and then treated with cytokines, or cytokines + SAFit2 (E). Relative proteins expression levels were quantified by Image J (F). Experiments were repeated for 3 times. Student’s t-test. Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The blotted membranes were blocked with 5% skim milk for 60min at room temperature and incubated with primary antibodies FKBP51 (1:1000, ABclonal, A3863, Woburn, MA, USA), p-FOXO1 (1:1000, NB100-81927, Novus Biological, Cambridge, UK), FOXO1 (1:1000, CST-2880, Cell signaling technology, Danvers, MA, USA), p-AKT(1:1000, CST4060S, Cell signaling technology, Danvers, MA, USA), AKT(1:1000, CST9272S, Cell signaling technology, Danvers, MA, USA), PDX1(1:1000, CST9679S, Cell signaling technology, Danvers, MA, USA), Sqstm1/p62 (1:1000, ab56416, Abcam, Cambridge, MA, USA), LC3I/II (1:1000, CST-12741S, Cell signaling technology, Danvers, MA, USA), BAX(1:1000, ab32503, Abcam, Cambridge, MA, USA), BCL2(1:1000, ab59348, Cambridge, MA, USA), β-actin (1:1000, CST-3700, Cell signaling technology, Danvers, MA, USA), Histone cluster 1, H3a (1:1000, 17168-1-AP, Proteintech, Rosemont, IL 60018, USA) at 4°C overnight.

Techniques: Inhibition, Western Blot, Transfection, Control, Expressing, Staining, Cytometry

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 6. Western analyses of content of Akt, pAkt, FoxO1, and pFoxO1 in aortic lysates from endothelial Nox4−/− and littermate mice fed the 0.3% and 4.0% NaCl diets. A, While on the 0.3% NaCl diet, the endothelial Nox4−/−mice demonstrated less Akt activity (pAkt(S473)) (P < 0.05) than the littermates. When fed the 4.0% NaCl diet, activation of Akt increased (P < 0.05) in the littermate controls. There were significant effects of Nox4 (P < 0.05) and dietary salt (P < 0.05) on Akt activation and an interaction effect (P < 0.05) between Nox4 and dietary salt. (n = 4 mice in each group; ns, not significant; *P < 0.05; Two- way ANOVA). B, Activation of FoxO1 (pFoxO1(S256)) and FoxO3a (pFoxO3a (S253)), two downstream phosphorylation targets of Akt [50,53], increased during the high salt intake. There were significant ef fects of Nox4 (P < 0.05) and dietary salt (P < 0.05) on FoxO1 and FoxO3a activation as well as an interaction effect (P < 0.05) be tween Nox4 and dietary salt on FoxO1 acti vation. (n = 4 mice in each group; ns, not significant; *P < 0.05; Two-way ANOVA).

Article Snippet: Experiments also used commercial antibodies directed against FoxO1 (Cat# NB100-2312, Novus Biologicals, Centennial CO); p-FoxO1 (ser256) (Cat# NB100-81927, Novus Biologicals), Runx2 (Cat# ab23981, Abcam Inc., Cambridge, MA), Runx2 (C-12) (Cat# sc-390715, Santa Cruz Biotechnology, Inc., Dallas, TX), PP2A (clone 1D6, Cat# 05–421, Millipore, Temecula, CA), osteopontin (Cat# ab283656, Abcam Inc., Cambridge, MA), osteocalcin (Cat# ab133612, Abcam Inc., Cambridge, MA), and GAPDH (Cat# Ab8245; Abcam Inc., Cambridge, MA), which served as a loading normalization control.

Techniques: Western Blot, Activity Assay, Activation Assay, Phospho-proteomics

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 7. Inhibition of Akt activation pre vented H2O2-induced increases in Runx2 and decreases in catalase in vascular smooth muscle cells (VSMC). A, Addition of LY294002, a pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) [37], inhibited the activation of Akt during over night incubation of VSMC in medium con taining H2O2, 0.4 mM. Both H2O2 (P < 0.05) and LY294002 (P < 0.05) affected Akt acti vation and an interaction effect (P < 0.05) between H2O2 and LY294002 on Akt acti vation was observed. (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA) B and C, Phosphorylated FoxO1 (pFoxO1(S256)) and FoxO3a (pFoxO3a(S253)), two downstream phos phorylation targets of Akt [50,53], increased during incubation of VSMC in medium con taining 0.4 mM H2O2. The addition of LY294002 inhibited these responses. There were significant effects of H2O2 (P < 0.05) and LY294002 (P < 0.05) on FoxO1 and FoxO3a activation as well as an interaction effect (P < 0.05) between H2O2 and LY294002 on FoxO1 activation (n = 4 sam ples in each group; ns, not significant; *P < 0.05; Two-way ANOVA). D, Addition of H2O2, 0.4 mM, into medium increased VSMC Runx2, which was prevented by LY294002. Both H2O2 (P < 0.05) and LY294002 (P < 0.05) affected Runx2 levels and an interac tion effect (P < 0.05) between H2O2 and LY294002 on Runx2 levels was observed (n = 4 samples in each group; ns, not signifi cant; *P < 0.05; Two-way ANOVA). E, Addition of H2O2, 0.4 mM, into medium of VSMC decreased catalase; the addition of LY294002 mitigated this decrease in protein levels. Both H2O2 (P < 0.05) and LY294002 (P < 0.05) affected catalase levels in VSMC and an interaction effect (P < 0.05) between H2O2 and LY294002 on catalase levels was observed (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA). F, Consistent with the western analysis data, H2O2, 0.4 mM, decreased catalase activity and this effect was miti gated by the addition of LY294002. Both H2O2 (P < 0.05) and LY294002 (P < 0.05) affected catalase activity, but there was no interaction effect (P = 0.1355) between H2O2 and LY294002 (n = 8 samples in each group; *P < 0.05; Two-way ANOVA).

Article Snippet: Experiments also used commercial antibodies directed against FoxO1 (Cat# NB100-2312, Novus Biologicals, Centennial CO); p-FoxO1 (ser256) (Cat# NB100-81927, Novus Biologicals), Runx2 (Cat# ab23981, Abcam Inc., Cambridge, MA), Runx2 (C-12) (Cat# sc-390715, Santa Cruz Biotechnology, Inc., Dallas, TX), PP2A (clone 1D6, Cat# 05–421, Millipore, Temecula, CA), osteopontin (Cat# ab283656, Abcam Inc., Cambridge, MA), osteocalcin (Cat# ab133612, Abcam Inc., Cambridge, MA), and GAPDH (Cat# Ab8245; Abcam Inc., Cambridge, MA), which served as a loading normalization control.

Techniques: Inhibition, Activation Assay, Incubation, Western Blot, Activity Assay

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 8. siRNA-mediated knockdown of FoxO1 or FoxO3a increased basal Runx2 levels and H2O2-induced increases in Runx2 in VSMC. A, Western blot showed successful knockdown of FoxO1 by FoxO1-specific siRNA. Addition of H2O2 into the medium induced the inhibition of FoxO1 (indicated by the increase in pFoxO1 (S256)). In these studies, FoxO1 siRNA (P < 0.05), but not H2O2 (P = 0.07), affected levels of pFoxO1(S253) in VSMC, but there was an interaction effect (P < 0.05) between H2O2 and FoxO1 siRNA on pFoxO1(S256). (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA) B, Knockdown of FoxO1 increased basal expression of Runx2 and increased H2O2-mediated increases in Runx2. H2O2 (P < 0.05) and FoxO1 siRNA (P < 0.05) affected levels of Runx2 in VSMC and an interaction effect (P < 0.05) between H2O2 and FoxO1-specific siRNA on levels of pFoxO1(S256) was observed. (n = 4 samples in each group; *P < 0.05; Two-way ANOVA) C, Western blot showed successful knockdown of FoxO3a by FoxO3a-specific siRNA. Addition of H2O2 into the medium induced the inhibition of FoxO3a (indicated by the increase in pFoxO3a(S253)). In these studies, H2O2 (P < 0.05) and FoxO3a siRNA (P < 0.05) affected levels of pFoxO3a(S253) in VSMC and an interaction effect (P < 0.05) between H2O2 and FoxO3a siRNA on pFoxO3a(S253) was observed (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA). D, Knockdown of FoxO3a increased basal expression of Runx2 and increased H2O2-mediated increases in Runx2. H2O2 (P < 0.05) and FoxO3a siRNA (P < 0.05) affected levels of Runx2 in VSMC, but an interaction effect (P > 0.05) between H2O2 and FoxO3a siRNA on levels of pFoxO3a(S253) was not observed (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA).

Article Snippet: Experiments also used commercial antibodies directed against FoxO1 (Cat# NB100-2312, Novus Biologicals, Centennial CO); p-FoxO1 (ser256) (Cat# NB100-81927, Novus Biologicals), Runx2 (Cat# ab23981, Abcam Inc., Cambridge, MA), Runx2 (C-12) (Cat# sc-390715, Santa Cruz Biotechnology, Inc., Dallas, TX), PP2A (clone 1D6, Cat# 05–421, Millipore, Temecula, CA), osteopontin (Cat# ab283656, Abcam Inc., Cambridge, MA), osteocalcin (Cat# ab133612, Abcam Inc., Cambridge, MA), and GAPDH (Cat# Ab8245; Abcam Inc., Cambridge, MA), which served as a loading normalization control.

Techniques: Knockdown, Western Blot, Inhibition, Expressing

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 11. Proposed redox-mediated endo thelial cell-aortic smooth muscle cell communication. When fed a diet contain ing 4.0% NaCl, endothelial activity of NOX4 produced amounts of H2O2 sufficient to generate an endothelial cell-aortic smooth muscle cell communication that directly altered smooth muscle function. Specifically, reduction in PP2a activity promoted Akt activation and downstream inactivation of FoxO1 and FoxO3a. Stimulation of this important signaling pathway increased Runx2 and reduced catalase, which facili tated the activity of H2O2 and increased further Runx2 in aortic smooth muscle.

Article Snippet: Experiments also used commercial antibodies directed against FoxO1 (Cat# NB100-2312, Novus Biologicals, Centennial CO); p-FoxO1 (ser256) (Cat# NB100-81927, Novus Biologicals), Runx2 (Cat# ab23981, Abcam Inc., Cambridge, MA), Runx2 (C-12) (Cat# sc-390715, Santa Cruz Biotechnology, Inc., Dallas, TX), PP2A (clone 1D6, Cat# 05–421, Millipore, Temecula, CA), osteopontin (Cat# ab283656, Abcam Inc., Cambridge, MA), osteocalcin (Cat# ab133612, Abcam Inc., Cambridge, MA), and GAPDH (Cat# Ab8245; Abcam Inc., Cambridge, MA), which served as a loading normalization control.

Techniques: Activity Assay, Produced, Activation Assay