Phosphorylated p38 p p38

Cultured BMECs were subjected to the Cell Lysis Buffer System (Santa Cruz, USA) supplemented with phenylmethylsulfonyl fluoride (PMSF, Santa Cruz, USA). A Cytoplasmic Extraction Kit (Beyotime, China) was used to extract the protein according to the manufacturer’s instructions. A BCA kit (Pierce, USA) was used to determine the protein concentrations of the samples, which were then subjected to the vertical SDS-PAGE. The separated proteins were then electronically transferred to PVDF membranes. Primary antibodies against Bax (Abcam, USA), Bcl-2 (Abcam, USA), activated caspase3 (Abcam, USA), p38 (Abcam, USA), phosphorylated p38 (p-p38, Abcam, USA), JNK (Abcam, USA), phosphorylated JNK (p-JNK, Abcam, USA), ERK1/2 (Abcam, USA), phosphorylated ERK1/2 (p-ERK1/2, Abcam, USA), and GAPDH (Abcam, USA) were used to incubate the membranes at 4°C for 8 h. The membranes were washed with TBST and then incubated with HRP-conjugated secondary antibodies. The membranes were developed by using an ECL kit (Pierce, USA). The densities of the immunoblots were determined and analyzed by Gene Genius (Syngene, England) and Image J (VER1.28, NIH, USA). Six independent experiments were carried out for immunoblots density quantification.

Western blot was performed using the soleus muscle. Proteins were electrophoresed on a 12% running gel. After electrophoresis, the gel was transferred to polyvinylidene diflouride membranes (Millipore, Bedford, MA, USA) at 4°C for 12 hr using a blotting device. Blocking was performed for 1 hr with 5% bovine serum albumin diluted with Tris-buffered saline in Tween-20 at room temperature. Primary antibodies against Akt, phosphorylated Akt (p-Akt), FoxO, phosphorylated FoxO (p-FoxO) (Cell Signaling, Beverly, MA, USA), MuRF1, p38, and phosphorylated p38 (p-p38) (Abcam, Cambridge, MA, USA) were prepared using 5% bovine serum at room temperature. The secondary antibody was reacted with horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) for 1 hr, respectively. Thereafter, each protein band was identified using an enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK).