Stat3 d3z2g

Western blotting was performed as previously described^{37 (link)}. The antibodies used in this study were E-cadherin (24E10, Cell Signaling Technology, USA), MMP9 (D6O3H, Cell Signaling Technology, USA), MMP2 (D4M2N Cell Signaling Technology, USA), vimentin (D21H3, Cell Signaling Technology, USA), PTEN (D4.3, Cell Signaling Technology, USA), p-PTEN (44A7, Cell Signaling Technology, USA), STAT3 (D3Z2G, Cell Signaling Technology, USA), p-STAT3 (D3A7, Cell Signaling Technology, USA), snail (C15D3, Cell Signaling Technology, USA), and GAPDH (D16H11, Cell Signaling Technology, USA). GAPDH was used as an internal control. The immunoreactive bands were visualized with ECL Ultra (New Cell and Molecular Biotech, Suzhou, China). All western blots were repeated three times with separate cell lysates and the statistical analysis were provided in the supplementary figures.

The IL-35 or IL-35 + IL-6 treated primary lung epithelial cells were lysed using the RIPA Lysis Buffer ordered from Beyotime (Shanghai, China) and added protease inhibitor cocktails freshly (Sigma-aldrich, St. Louis, USA). Total protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The expression level of target proteins was determined by Western blot as previously described [20 (link)]. Antibodies used in this study were listed below: rabbit monoclonal antibody for phospho-Stat1 (Tyr701) (D4A7, 1:1000 dilution), Stat1 (D1K9Y, 1:1000 dilution), phospho-Stat3 (Tyr705) (D3A7, 1:1000 dilution), Stat3 (D3Z2G, 1:1000 dilution) and Stat4 (C46B10, 1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, USA). Antibody for p-Stat4 (E-2) (sc-28296, 1:1000 dilution) was ordered from Santa Cruz Biotechnology, Inc. (Dallas, USA).

Cells were lysed in RIPA lysis buffer containing 50 mm HEPES, 150 mm NaCl, 1 mm EDTA, 1% (w/v) CHAPS, and protease and phosphatase inhibitor cocktail (Sigma‐Aldrich, St. Louis, MO, USA). Subsequently, the cell lysates were boiled in 2× SDS/PAGE loading buffer for 10 min, and then, 25 μg of protein was separated on a 4–20% SDS/PAGE and transferred to Hybond‐C Extra nitrocellulose membrane. Primary antibodies used in the experiments were β‐actin (ab8226; Abcam, Eugene, OR, USA), ERα (M7047; DAKO), HIF‐1α (#61095,9; BD Biosciences, San Jose, CA, USA), Phospho‐Stat3 (Tyr705; 9131; Cell Signalling), STAT3 (D3Z2G; Cell Signalling, Danvers, MA, USA), and PR (M3569; DAKO, Agilent, Santa Clara, CA, USA). HRP‐conjugated secondary antibodies were purchased from Life Technologies. Protein detection was carried out using ECL system (GE Healthcare, Chicago, IL, USA).