Il10ra

Cultured fibroblasts, with a 70%‐80% confluence after incubation for 12‐16 hours in serum‐free medium, were stimulated with LPS (l‐2880, 1.0 μg/mL, Sigma), IL‐10 (#200‐10, 10 ng/mL, PeproTech), IL10RA (sc‐365374, 1:500, Santa Cruz) or cryptotanshinone (s2285, 4.6 μmol/L, Selleckchem) for 48 hours. As for the inhibitory role of IL‐10 signalling, siRNAs for IL10Rɑ (NM001558.3) were also used, siIL10Rɑ sense: 5′‐gucugaaaguaccugcuauga‐3′, anti‐sence: 5′‐ucauagcagguacuuucagac‐3′. Using 50 nmol/L siRNA fragment, the transfection incubation time for siRNA/Lipofectamine RNAiMAX reagent complexes was 48 hours, then washed in PBS and resuspended in RIPA cell lysis solution (Beyotime) supplemented with 200 μg/mL phenylmethylsulfonyl fluoride (PMSF, Boster), phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma). The BCA assay (Pierce) was used to determine the protein concentration of the cell lysate. Then, the Western blotting was performed as previously described.²³, ³⁹, ⁴⁰