Af4198

PMA was obtained from Sigma (P1585). Anti-COX-2 and anti-CD68 antibodies were purchased from R&D Systems (AF4198 and MAB20401). Rabbit polyclonal anti-RGC-32 antibody was purchased from Biorbyt (orb2372). Anti-smad2 and anti-p-smad2 were purchased from Cell Signaling Technology (5339 and 18338). TGF-β1 neutralizing antibody (NAb) was obtained from R&D Systems (MAB240) and used at 2.5 μg/ml.

After stimulation, cells were lysed for 10 min in ice using buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM NaVO₄, 20 mM PMSF, Phosphatase inhibitor cocktail 3 (1 in 100 dilution, Sigma) and Protease inhibitor cocktail (1 in 100 dilution, P8340, Sigma). Protein levels were quantified using Pierce BCA Protein Assay Kit (Life Technologies) and adjusted to 500 μg/mL for immunoblotting. The samples were then incubated for 5 min at 100°C with Pierce Lane Marker Reducing Sample Buffer (Life Technologies). Gels were loaded with 20 μL of the sample per lane, with a final protein mass of 10 μg.
Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1β (goat) (AF-401, R&D Systems) 1 in 1000; β-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems). The secondary antibodies used were: anti-goat IgG-HRP (sc-2922, Santa Cruz) 1 in 5000; anti-mouse IgG-HRP (7076, Cell Signaling) 1 in 6000; anti-rabbit IgG-HRP (A24537, Thermo Scientific) 1 in 6000 as appropriate; anti-rat IgG-HRP (7077S, Cell Signaling) 1 in 5000.