Phospho-FOXO3A (Ser413) Antibody - #AF2343

Figure 6 RES treatment promotes the activation of FoxO3a, which is mediated by SIRT1 and AMPK. (A) BMDMs from mSirt1+/+ and mSirt1−/− mice were infected with the RH strain (MOI = 1, for 2 h) and then further stimulated with RES (10 µM) for 18 h. Cells were fixed and immunostained with an anti-FoxO3 antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, green) and DAPI for nuclei (blue). Cells were then subjected to immunofluorescence microscopy. Representative images (left) and quantitative data (right) showing the cytoplasmic translocation of FoxO3a were obtained from one representative of 3 independent samples, with each experiment containing a minimum of 50 cells scored in 7 random fields. Scale bar = 25 µm. (B) BMDMs from mSirt1+/+ and mSirt1−/− mice were stimulated with RES (10 µM) for the indicated time periods. The phosphorylation of FoxO3 (Ser413) was determined by immunoblot analysis. (C) BMDMs were infected with T. gondii for 18 h in the presence of RES (10 µM) or EX527 (4 µM). Cell lysates were subjected to immunoprecipitation analysis using an anti-acetylated-lysine antibody (Ac-Lys), and then the immunoprecipitates were analysed by Western blot using an anti-FoxO3a antibody. Cell lysates were harvested and subjected to Western blot analysis for FoxO3a and β-tubulin, as loading controls. (D) BMDMs were pretreated with Compound C (25 µM) or STO-609 (10 µM) for 45 min, then infected with RH strain (MOI = 1) for 18 h in the presence or absence of RES (10 µM). Cells were fixed, immunostained, and quantified as described in Figure 6A. Representative images (left) and quantitative data (right) showing the cytoplasmic translocation of FoxO3a. Scale bar = 25 µm. (E) BMDMs were pretreated with Compound C (25 µM) or STO-609 (10 µM, 45 min) and then stimulated with RES (10 µM, 18 h). The phosphorylation of FoxO3 (Ser413) was determined by immunoblot analysis. Data are representative of three independent experiments and are presented as means ± SD. *** p < 0.001 (two-tailed Student’s t-test). Tg, Toxoplasma gondii; RES, resveratrol; CompC, Compound C; STO, STO-609; ns, not significant.

FIGURE 6|Effects of simvastatin on the AMPK-SKP2-CARM1 pathway in corpus cavernosum of DMED rats. (a) Representative Western blotting results of p-AMPK, AMPK, p-FoxO3a, SKP2, CARM1, TFEB, and TBP in the corpus cavernosum of the control, DMED, and DMED + sim group.