Chemiscope 6200 chemiluminescence imaging system

The effects of abdominal massage on the PI3K-Akt signal pathway were investigated by western blot. All proteins were extracted using a mammalian protein extraction kit (Sangon, China), and the protein concentration was detected by the BCA method (Pierce, USA). The 30 mg protein was electroblotted onto a polyvinylidene fluoride (PVDF) membrane following separation on an 8% SDS-polyacrylamide gel. The PVDF membrane was incubated with blocking solution (5% skim milk) for 2 h and overnight with a 1 : 1,000 dilution of anti-GDNF, anti-P-Akt, and anti-GAPDH antibodies at 4°C. Blots were washed and incubated with a 1 : 2,000 dilution of the horseradish peroxidase-conjugated secondary antibody. Blots were washed five times with TTBS again and then developed using a horseradish peroxidase substrate and captured on a ChemiScope 6200 chemiluminescence imaging system (Clinx Science Instruments Co., Ltd.).

Cells were washed in cold PBS and lysed in protein lysis buffer (Keygentec, KGP1100) for 0.5 h at 4 °C. The supernatant was collected after centrifuging for 10 min at 12 000 × g. Protein concentration was measured by the BCA Protein Assay kit (Keygentec, KGP903). Proteins were separated on ≈10–12% SDS‐PAGE gels. Following incubation with primary antibody and HRP conjugated secondary antibodies, signals were captured using ChemiScope 6200 Chemiluminescence imaging system (CLINX). The following antibodies were used, rabbit anti‐ATF4 (1:500, Santa, SC‐200), rabbit anti‐p‐S6 (1:1000, CST, 4856S), rabbit anti‐S6 (1:1000, CST, 2217S), rabbit anti‐β‐actin (1:1000, Proteintech, 20536‐1‐AP), rabbit anti‐β‐tubulin (1:1000, CST, 2146S), rabbit anti‐NNMT (1:1000, Proteintech, 15123‐1‐AP), and rabbit anti‐DNMT1 (1:1000, CST, 5032).