Py701 stat1

HepG2 cells were seeded in a 6-well plate at a density of 5 × 10⁵ ml^–1 in complete medium and cultured for 12 h. Cells were then pre-treated with compounds (in 0.05% DMSO) or vehicle control in low-FBS medium for 6 h. IL-6 (25 ng/ml) was added to the wells to induce the phosphorylation of STAT3.^{63 (link), 64 (link)} After 30 min, cells were harvested and protein samples were quantified by the Pierce BCA Protein Assay Kit. 30 μg of each protein sample was separated by SDS-PAGE. In the IFN-α-induced comparative assay, HeLa cells were seeded in a 6-well plate at a density of 5 × 10⁵ ml^–1 in complete medium and cultured for 12 h. Cells were then pre-treated with compounds (in 0.05% DMSO) or vehicle control in low-FBS medium for 6 h. IFN-α (1000 U/ml) was added to the wells to induce the phosphorylation of STAT3 and STAT1. After 1 h, cells were harvested and proteins samples were quantified. 30 μg of each protein sample was separated by SDS-PAGE. The proteins were transferred to a PVDF membrane, and blocked with 5% non-fat dry milk in TBST for 1 h at room temperature. The membranes were incubated with primary antibodies (pY705-STAT3, pY701-STAT1, STAT3, STAT1 and GAPDH antibodies, Cell Signaling Technology) overnight. After treatment with secondary antibodies, membranes were analyzed with enhanced chemiluminescent Plus reagents (GE Healthcare).

Sorted MZ B cells (5 × 10⁵) were left untreated or treated with 10 µg/ml LPS, 10 µg/ml CpG ODN 1826, or 1,000 U IFN-α4 for 1 h. Cells were lysed in IP lysis buffer (300 mM NaCl, 50 mM Hepes, pH 7.6, 1.5 mM MgCl₂, 10% glycerol, 1% Triton X-100, 10 mM NaPyrPO4, 20 mM NaF, 1 mM EGTA, and 0.1 mM EDTA) plus freshly added 1 mM DTT, 1 mM PMSF, 10 µl proteinase inhibitor cocktail (Sigma-Aldrich), 1 mM Na₃VO₄, and 20 mM NaF. Primary antibodies pS727-STAT1 (Cell Signaling Technology) and pY701-STAT1 (Cell Signaling Technology) and STAT1 (made in-house) were used.