Anti ep4

Immunohistochemical studies were performed using a rabbit polyclonal antibody against mPGES-1 (ref. HPA045064 prestige antibodies, diluted 1 : 50) from Sigma and a mouse monoclonal antibody anti-EP-4 (ref. 101775, diluted 1 : 100) from Cayman Chemical. Blanks were performed using the corresponding blocking peptides all from Cayman. Monoclonal antibodies (ref. M0616, diluted 1 : 35; ref. IR751 and ref. IR613, without further dilution) from Dako were used for von Willebrand Factor (vWF, endothelial cell marker), CD45 (pan-leukocyte marker), and CD68 immunostaining. Three-micrometer sections of paraffin-embedded tissue samples were stained in a Dako Autostainer Link 48 using the Dako EnVision Flex kit. Diaminobenzidine was used as chromogen. Immunostainings used for comparative purposes were processed simultaneously.

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).