Py705 stat3

Total protein was extracted from the lung tissues in mice, and the total protein and nucleoprotein were extracted from the PMVECs. The resultant protein concentrations were determined by BCA Protein Assay reagents (Beyotime Biotechnology, Jiangsu, China) according to the previous description^{18 (link)}. The expression of STAT3, pY705-STAT3, and Bcl-2 was determined using a standard protocol. The transferred membrane was blocked with 10% skimmed milk for 1 h at room temperature, and then incubated with the primary antibodies against STAT3 (1:1000; Abcam), and pY705-STAT3 (1:700; Abcam), Bcl-2 (1:700; Santa Cruz), and β-actin (1:500; Santa Cruz) overnight at 4 °C, respectively. After incubating with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Zhong Shan-Golden Bridge Biological Technology Company, Beijing, China) for 1 h at room temperature, the immunoblotting signals were visualized using a Western Luminescent Detection kit (Vigorous Biotechnology, Beijing, China).

GICs were lysed at 4°C for 30 min in RIPA buffer (Sigma Aldrich, St Louis, MO) supplemented with protease inhibitor (Sigma Aldrich, St Louis, MO) and phosphatase inhibitor (BioTools, Jupiter, FL), followed by centrifugation (10,000xg for 10 min). Protein extracts (50μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA), and immunoblotted with the following antibodies: STAT3 and pS727-STAT3 (BD Biosciences, San Jose, CA), pY705-STAT3 (Abcam, Cambridge, A), STAT1 and STAT5 (Cell Signaling, Danvers, MA), and Actin (Santa Cruz Biotechnology, Dallas, TX). Following addition of IRDye800CW goat anti-mouse IgG or IRDye680 goat anti-rabbit IgG, blots were visualized on an Odyssey infrared imaging system (LICOR Biosciences, Lincoln, NE).

Total/cytoplasm/nucleus protein was extracted from tumor tissues, non-tumor adjacent tissues, or liver cancer cell lines using the Total/Cytoplasm/Nucleus Protein Extraction Kit (Solarbio, China). An equivalent of 50 µg of the protein extract was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane was blocked for 2 h at room temperature using milk (5%) was used to block membranes. Subsequently, the membranes were probed with primary antibodies, including rabbit polyclonal antibodies to AQP3 (1:2000, Abcam, USA), CD133 (1:1500, Abcam), JAK1 (1:1000, Abcam), pY-JAK1 (1:2000, Abcam), JAK2 (1:1500, Abcam), pY-JAK2 (1:1500, Abcam), STAT3 (1:2000, Abcam), pY⁷⁰⁵-STAT3 (1:2000, Abcam), GAPDH mouse monoclonal antibody (1:2000, Abcam), Histone H3 (phospho S10) rabbit monoclonal antibody (1:500, Abcam) overnight at 4 °C, followed by incubation with secondary antibodies for 2 h at room temperature. The immunoreactive bands were identified using an ECL system (Millipore, USA). Every tissue was evaluated three times using Western blotting.