0.45 μm pvdf membrane

Cell lysate (cat. no. P0013J, Beyotime, Shanghai, China) lysed 8.5 × 10⁷ BMSCs in each group and centrifuged at 4 °C and 12000 rpm for 10 min. Protein concentrations were measured by a BCA protein concentration detection kit (cat. no. P0011, Beyotime, China), 55 μg of BMSC lysate samples from each group were subjected to SDS–PAGE on a 0.45 μm PVDF membrane (cat. no. FFP36, Beyotime, China). After the skimmed milk powder was sealed for 60 min, rabbit anti-human phospho Akt antibody (P-Akt, cat. no. ab8933, 1:250, Abcam, Cambridge, USA) and rabbit anti-human Akt antibody (cat. no. ab18785, 1:200, Abcam, USA) and incubated at 4 °C for 18 h; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (cat. no. ab6721, 1:500, Abcam, USA) was incubated for 240 min at room temperature. β-actin (cat. no. ab5964, 1:400, Abcam, USA) was the internal reference control. An ultrasensitive ECL chemiluminescence kit (cat. no. P0018AS, Beyotime, Shanghai, China) and a Tanon 1600 gel image analysis system (Tanon. ltd., Shanghai, China) were used to detect protein expression. Image-Pro Plus 6.0 (Mediacy Cybernetics, Inc. Bethesda, MD, USA) analyzes the expression of each protein band.