Western Blotting Analysis of Cellular Signaling

Western blot analyses were performed as using the following method^{9 (link),46 (link)}. Briefly, cells were lysed and scraped in RIPA buffer (Thermo Scientific, IL, USA). The following primary antibodies were used for immunoblotting: anti-CaMKK2, anti-phospho-CaMKK2, anti-AMPK, and anti-phospho-AMPK, anti-MLC and anti-phospho-MLC,which were obtained from CST (Cell Signaling Technology, MA, USA); anti-GAPDH, which was obtained from Santa Cruz Biotechnology (CA, USA); anti-EP4, which was obtained from Cayman (MI, USA); anti-CALML6, which was obtained from Proteintech (IL, USA); and anti-PGC1-α, which was obtained from Abcam (Cambridge, UK). Chemiluminescence detection was performed using ECL reagent (Bio-Rad Laboratories, CA, USA) and high-sensitivity ECL reagent (Thermo Scientific, IL, USA). Signals were visualized using a LuminoGraph II imaging system (ATTO, Tokyo, Japan). The signal intensities of the bands were quantified using ATTO CS Analyzer 4 software (ATTO).