Ptgs2

Protein was isolated with 100 µL of CelLytic (C2978, Sigma Aldrich, St. Louis, MO, USA) supplemented with proteinase inhibitor (87786, Thermo Fisher Scientific, Waltham, MA, USA) and concentration was determined with RotiQuant (K015.3, Carl Roth, Karlsruhe, Germany) according to manufacture’s instructions. Equal amounts of protein were separated on 10% polyacrylamid gels. The proteins were transferred to a PVDF membrane with a pore size of 0.45 µm (T830.1, Carl Roth, Karlsruhe, Germany) for 90 min at 90 V. To prevent subsequent nonspecific binding of antibodies to the membrane, the blotted membrane was incubated for 1 h at room temperature in 5% milk powder (T145.3, Carl Roth, Karlsruhe, Germany) dissolved in Tris-buffered saline with Tween20 (TBS-T). ACTIN (E1C602-2, EnoGene, New York, NY, USA) and PTGS2 (PA5-16817, Thermo Fisher Scientific, Waltham, MA, USA) were used as primary antibodies. These were each incubated overnight at 4 °C and after washing three times in TBS-T, the membrane was incubated in the secondary antibody (611-1302, Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h at room temperature. After washing in TBS-T, Luminata Crescendo Western HRP Substrate (WBLUR0100, Sigma Aldrich, St. Louis, MO, USA) was added and the signal was digitized using the VWR Genoplex documentation system (VWR international, Radnor, PA, USA).