Protease inhibitor cocktail mixture

Immunoprecipitation (IP) was performed using Pierce Crosslink Immunoprecipitation Kit (Thermo, 26147) following the manufacturer’s protocol. Cells were lysed in RIPA lysis buffer (25 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol) with the presence of a protease inhibitor cocktail mixture (Sigma-Aldrich). Cells lysates were pre-cleared by the control agarose resin and then immunoprecipitated using anti-STC2 antibody (Abcam, ab255610) overnight at 4 °C. Antigen was eluted and subjected to SDS page electrophoresis.

The tissues were homogenized and lysed in a buffer containing 40 mmol/L Tris/HCl (pH 7.4), 150 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L dithiothreitol, 1% Triton X-100, 2 mmol/L sodium orthovanadate, 10 mmol/L NaF, and 10 mmol/L sodium pyrophosphate supplemented with a protease inhibitor cocktail mixture (Sigma, St Louis, MO, USA). Protein contents were measured by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and bovine serum albumin was used as a standard. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then proteins were transferred onto an Amersham Hybond PVDF membrane (GE Healthcare, Little Chalfont, UK). After a blocking procedure, the membrane was incubated with anti-mPGES-1 (No. 160140; Cayman Chemicals, Ann Arbor, MI, USA), anti-cPGES (No. 160150; Cayman Chemicals), anti-COX-2 (No. 160106; Cayman Chemicals), anti-COX-1 (NAB37401; R&D Systems, Minneapolis, MN, USA), or anti-β-actin (clone 2F3; Fujifilm Wako Pure Chemical, Osaka, Japan) antibody and then incubated with a secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories, PA, USA). After washing, protein was detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).