STAT3 ELISA

NHLF cells (2×10⁵ cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS. Positive control response to IL-6 was determined by adding 50 ng/ml of IL-6 (R&D) to the cells and incubating for 60 min followed by measuring STAT3 phosphorylation. STAT3 phosphorylation at tyrosine residue 705 was examined in cell lysates using STAT3 (pY705) ELISA kit (Invitrogen, KHO0481) according to the manufacturer's protocol. Extracellular IL-6 receptor activation was blocked using anti-IL-6r (30 mg/ml, R&D) or anti-gp130 (30 mg/ml, R&D) at 37°C for 30 min prior incubation with IL-6, or for 30 min on 1 kPa or tissue culture plastic. To test roles in STAT3 phosphorylation, inhibitors of ROCK (Y27632, 3-30 µM, STEMCELL Technologies), myosin II (Blebbistatin 3-30 µM, Selleckchem), JAK1 (Ruxolitinib, 1-10 µM, Selleckchem), JAK2 (AZD1480, 1-10 µM, Selleckchem), JAK3 (Tofacitinib, 1-10 µM, Selleckchem), TGF-β type I receptor (1-10 µM, Selleckchem) and FAK (PF562271, 1-10 µM, Selleckchem) were added to the cells and incubated for 1 h prior to lysis and measurement of STAT3 phosphorylation.