Cells (1 × 105–4 × 105 cells/well) were seeded in 6-well plates and treated with indicated condition. Then, the cells were lysed and incubated on ice for 30 min. After being centrifuged at 12,000 rpm for 15 min, the protein was separated by electrophoresis on 12% SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% skimmed milk and incubated with indicated primary antibodies. The incubated mixture was washed with TBST. Then horseradish peroxidase (HRP) secondary antibodies were added with the mixture incubating at 37 °C for 2 h. The incubated mixture was washed with TBST. HRP electrogenerated chemiluminescence (ECL) was used to develop, the developed films were taken and rinsed with pure water, and the washed films were dried. Scanning was used for recording.
cAMP levels were measured by the cAMP Direct Immunoassay Kit (Abcam, Cambridge, MA, USA) following Abcam’s protocol. PKA activity was evaluated by the PKA Kinase Activity Assay Kit (Abcam, Cambridge, MA, USA) and following Abcam’s protocol. Briefly, after treatment, the protein content of each sample was determined with Bradford assay; 10 µg of each lysates was used for detecting optical density at 450 nm by ELISA method.
cAMP levels were measured by the cAMP Direct Immunoassay Kit (Abcam, Cambridge, MA, USA) following Abcam’s protocol. PKA activity was evaluated by the PKA Kinase Activity Assay Kit (Abcam, Cambridge, MA, USA) and following Abcam’s protocol. Briefly, after treatment, the protein content of each sample was determined with Bradford assay; 10 µg of each lysates was used for detecting optical density at 450 nm by ELISA method.