Ac foxo1

Paraffin sections were washed with a gradient series, rinsed with distilled water, and subjected to antigen retrieval, quenching autofluorescence, and bovine serum albumin blocking. The sections were then incubated with Laminin (Cat. No. ab207612, Abcam, UK), FoxO1 (Cat. No. ab52857, Abcam, UK), Ac‐FoxO1 (Cat. No. Orb543702, Biorbyt, UK) and Myosin antibodies (Cat. No. ab124205, Abcam, UK) overnight at 4°C, followed by secondary antibodies (Cat. No. GB21303/926–32,210, Servicebio, China) after washing with buffer at room temperature for 50 min. Cell nuclei were counterstained with DAPI, mounted onto slides, and photographed under a microscope. Five different fields of view were selected from the centre and four corners of each slice, and Image pro Plus 6.0 (Media Cybernetics, Inc. USA) software was used for blind analysis by a third person. The FCSA of muscle were calculated, and the integrated optical density (IOD) value of the selected area was measured to indicate immunopositivity. The percentages of FoxO1 and Ac‐FoxO1 nuclear localisation were calculated.

To assess the protein levels of Bcl-2, cleaved-caspase-3, Ac-FoxO1, Ac-NF-κB, and Ac-p53, 50 μg of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk at room temperature for 3 h, incubated with primary antibodies specific to Bcl-2 (1:1000, Abcam, Cambridge, UK), cleaved-caspase-3 (1:1000, Abcam), Ac-FoxO1 (1:1000, Abcam), Ac-NF-κB (1:1000, Cell Signaling Technology, Beverly, MA), Ac-p53 (1:1000, Cell Signaling Technology), BAX (1:1000, Cell Signaling Technology), SIRT1 (1:1000, Cell Signaling Technology), or β-actin (1:1000, Abcam) at 4 °C overnight. Then, membranes were incubated with HRP-conjugated secondary antibodies diluted at 1:3000 (Boster, Wuhan, China) at 37 °C for 1 h. Protein bands on the membrane were visualized with ECL Kit (Millipore, USA) using FluorChem FC system (Alpha Innotech). Results were presented as densitometric ratio between the protein of interest and the loading control (β-actin).