Anti phospho stat1

Total protein was isolated from the lung tissue cleared of blood using RIPA buffer (Beyotime) containing protease (Beyotime) and phosphatase (Roche, Basel, Switzerland) inhibitors. The Pierce BCA Protein Assay Kit (Thermo Scientific, USA) was used to estimate the protein concentration. Lysates were adjusted to a concentration of 3 μg/μL. The protein samples were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (Millipore, Germany). The membranes were blocked with 5% skim milk for 1 h at room temperature in TBST buffer and probed with the following primary antibodies: anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3 (Affinity, 1 : 500), and anti-β-actin (CST, 1 : 1000) overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (CST, 1 : 2000). Protein bands were visualized using enhanced chemiluminescence (ECL). β-Actin was used as a loading control. ImageJ software was used to analyze the optical density of the protein bands.

According to the molecular weight of the target protein, cells or tissue samples containing the same amount of protein were loaded onto appropriate concentration SDS-PAGE for electrophoretic separation, and subsequently transferred to the PVDF membrane (Millipore, Billerica, MA, USA), which was then blocked in the blocking buffer (Epizyme, Shanghai, China) for 1 h at room temperature, followed by incubation overnight at 4 °C with the following primary antibodies: anti-β-actin (AC038, Abclonal, Wuhan, China), anti-STAT1 (AF6300, Affinity Biosciences, Changzhou, China), anti-phospho-STAT1 (AF3300, Affinity), anti-STAT3 (AF6294, Affinity), anti-phospho-STAT3 (AF3293, Affinity), anti-Bcl2 (A0208, Abclonal), anti-Cleaved-Caspase3 (9664S, Cell Signaling Technology), anti-GFAP (GB12096, Servicebio), anti-MAP2 (A0453, Abclonal), anti-Bax (A19654, Abclonal), anti-iNOS (13120S, Cell Signaling Technology), and anti-Arg1 (93668S, Cell Signaling Technology). On the following day, the membrane was incubated with relevant HRP-conjugated secondary antibody (S0001, S0002, Affinity) for 2 h, and visualized using ECL reagent (SD6032, Simuwu, Shanghai, China). The protein band images were obtained on the Chemiluminescent Imaging System (Tanon, 5200, China). All experiments were repeated three times. The density of protein bands was semi quantitatively analyzed by ImageJ software.