Anti foxo3a antibody

The indicated cells were cultured onto coverslips in 6-well plates until 70% confluence. Cells were then fixed with 4.0% paraformaldehyde in phosphate-buffered saline for 15 min, and blocked with 5% goat serum for 30 min. Next, the coverslips were incubated at 4°C with the indicated primary antibodies overnight. Anti-FOXO3a antibody was purchased from Cell Signaling Technology, Inc. Anti-E-cadherin and anti-Vimentin antibodies were purchased from Epitomics, Inc. Anti-N-cadherin antibody was purchased from Abcam, Inc. Subsequently, the coverslips were incubated with FITC-conjugated goat anti-rabbit secondary antibody (Abcam) or Cy3-conjugated goat anti-rabbit secondary antibody (Bioss, Beijing, P.R. China), dyed with Hoechst33342, and fixed in glycerol. The images were obtained with an Olympus IX71 microscope (Olympus, Tokyo, Japan), and color mergence was performed using ImageJ image software (ImageJ version 1.44p, NIH, MD).

The brains of mice were fixed with 4% paraformaldehyde and embedded in paraffin and then cut into sections with thickness of 4 μm using a paraffin slicer. Slices were boiled in a 10 mM citrate buffer (pH 6.0) for 10 min to expose antigens and then cooled to room temperature. The endogenous peroxides of sections were quenched with 0.3% H₂O₂ for 10 min. Slices were blocked with 5% normal goat serum for 10 min and then incubated with anti-BDNF antibody (1 : 200; Abcam, Cambridge, UK), anti-p-ERK1/2 antibody (1 : 200; Cell Signaling Technology, MA, USA), anti-p-CREB antibody (1 : 200; Cell Signaling Technology, MA, USA), anti-AKT antibody (1 : 200; Cell Signaling Technology, MA, USA), or anti-FOXO3a antibody (1 : 200; Cell Signaling Technology, MA, USA) overnight at 4°C. The resulting slides were washed and incubated with biotinylated secondary antibody (1 : 300; Proteintech, Chicago, USA) for 2 h at room temperature. Then, sections were stained with 3,3-diaminobenzidine (DAB, Zhongshan Jinqiao, Beijing, China) for 1-2 min, counterstained with hematoxylin for 20 s, decolorized, and sealed. Finally, the hippocampal subfields (CA1 and hilus) were observed under a 400x light microscope (Nikon Instruments Inc., Tokyo, Japan) and analyzed using ImageJ software.

For evaluating subcellular distribution of FoxO3a, NRCMs were permeabilized with 0.5% Triton X-100 in Tris-buffered saline with Tween 20 (0.5% TBST) and blocked with 2% bovine serum albumin in 0.1% TBST. NRCMs were then incubated with anti-FoxO3a antibody (1:100; Cell Signaling #12829) for 2 h at room temperature. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated anti-Rabbit secondary antibody (Thermo Fisher Scientific) for 1 h at room temperature. Then, cells were mounted with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were obtained using BZ-X700 fluorescence microscope (Keyence).

HEK293T cells were plated in 96-well plates and treated as indicated. Cells were then fixed in a final concentration of 3.7% formaldehyde by addition of fixative directly to the medium for 10min, then permeabilised for 10 min in PBS containing 0.1% Triton X-100. Cells were then incubated with anti-FOXO3a antibody (Cell Signaling Technology 12,829), diluted 1:1000 in PBS +1% bovine serum albumin (BSA) for 2hrs. Cells were washed in PBS + 1% BSA then incubated with Alexa Fluor 488 conjugated goat anti-rabbit IgG (A11034, Thermo Fisher Scientific) and 4 μg/ml Hoechst 33,342 (Invitrogen) for 1hr. Images were acquired on the ImageXpress Micro Confocal and analyzed as described above for screening.

Whole-cell protein was obtained for quantitative capillary isoelectric immunoassay. Protein levels were assessed using a capillary-based automated electrophoresis immunoquantification instrument (ProteinSimple, San Jose, CA, United States) following the manufacturer’s standard instruction. Here, 3 μl of protein extract (final concentration: 1 μg/μl) was loaded with the following antibodies: anti-SIRT1 antibody (sc15404, Santa Cruz Biotechnology, Santa Cruz, CA, United States), anti-GAPDH antibody (10494-1-AP, ProteinTech), and anti-FOXO3a antibody (12829, Cell Signal Technology, Inc., United States). The run conditions were as recommended previously. Compass software (ProteinSimple ver. 3.1.8) was utilized to calculate and measure the immunoblots.