Anti mpges 1

DMEM-F12, newborn bovine serum, HEPES and penicillin/streptomycin were purchased from Wisent Corporation (Wisent, Canada). Hank's balanced salt solution (HBSS), nonessential amino acids, sodium pyruvate, insulin-transferrin-selenium and L-glutamine were purchased from Invitrogen Life Technologies (Paisley, Scotland). Stainless steel sieves were obtained from Merck Eurolab (Leuven, Belgium). Anti-NKCC2 (Stressmarq Biosciences Inc., Canada), anti-NLRP3 (Abcam, Cambridge, MA), anti-mPGES1 and anti-COX2 (Cayman, Ann Arbor, MI) primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used.

Where indicated, cells were treated with RAR antagonist AGN193109 (5758, Tocris Bioscience, Bristol, UK) and RXR antagonist UVI3003 (3303, Tocris Bioscience) for 48 h (1 µM).
Total protein lysates were obtained using celLytic MT (C2978-50 mL, Sigma Aldrich), a cell lysis reagent, as described [32 (link)]. Antibodies used are as follows: anti-Oct-4A (2840 Cell Signaling, Milan, Italy), anti-Sox2 (3579 Cell Signaling), anti-KLF4 (4038 Cell Signaling), anti-Nanog (4903 Cell Signaling), anti-c-MYC (5605 Cell Signaling), anti-PD-L1 (13684; Cell Signaling); anti-ALDH3A1 (TA332730, Origene); anti-mPGES1 (160140) and anti-COX-2 (160112, Cayman Chemical, Arcore, Italy); anti-CD133 (PA1217, Boster), anti-NFkB (sc-372, Santa Cruz, CA, USA), and anti-β-actin (Sigma Aldrich). Images were digitalized with CHEMI DOC Quantity One program (Biorad, Milan, Italy

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).