Phospho-FOXO1A (Ser249) Antibody - #AF8271

Figure 5DOX induced CDK7-dependent activation of the proapoptotic CDK2–FOXO1–Bim axis. (A) C57BL/6 mice received a single injection of DOX (5 mg/kg, i.p.) and the CDK7 inhibitor THZ1 (10 mg/kg, twice daily) or carrier (n = 6 mice per group). Hearts were collected at 24 h after DOX injection. Protein levels were measured by western blotting. Two-way ANOVA with Tukey’s test. **P < 0.01. (B–G) NRCMs were pre-treated with the CDK7 inhibitor THZ1 (100 nM) for 1 h or transfected with control (siControl) or CDK7 siRNA (siCDK7, 50 nM) for 96 h prior to incubation with DOX (1 µM) for 4 h (n = 3). Two-tailed Student’s t-test. *P < 0.05, **P < 0.01. (B and E) Protein levels were measured by western blotting. (C and F) Bim mRNA levels were evaluated using semiquantitative RT–PCR. (D and G) Bim mRNA levels were evaluated using quantitative RT–PCR. (H) NRCMs were infected with GFP (adGFP) or human CDK7 adenovirus (adCDK7) for 96 h prior to treatment with DOX (1 µM) for 8 h (n = 3). Protein levels were measured by western blotting. Two-tailed Student’s t-test. *P < 0.05. (I) H9c2 cells were transfected with the Bim promoter construct Bim-LUC or Bim-LUC (dm) with mutations in the FOXO1-binding sites. Cells were then transfected with siCDK7, treated with THZ1, or infected with adCDK7 followed by incubation with DOX (1 µM) for 8 h (n = 4). Cells transfected with scrambled siRNAs, treated with vehicle, and infected with adGFP served as control. One-way ANOVA with Tukey’s test. *P < 0.05, **P < 0.01. The n value represented the number of individual mouse or individual cell culture well biological replicate.

Figure 2. |Stimulation with DOX enhanced FOXO1 phosphorylation by CDK2.(F) NRCMs transfected with HA-CDK2 were subjected to immunofluorescence staining for phospho-FOXO1 (S249, green), HA tag (red) and nuclei (DAPI, blue). Intense phospho-FOXO1 (S249) signal was observed in HA-positive cells (arrowhead), but not in HAnegative cells (arrows). Scale bar=20µm.