Anti p jak1

Spleen proteins were extracted using RIPA lysis buffer (Solarbio, Beijing, China) and centrifuged at 12,000 g for 10 min at 4°C. Protein concentration was assessed using the BCA Protein Assay Kit (Biosharp, Beijing, China). Samples containing equal amounts of protein (80 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to 0.45 μm polyvinylidene difluoride membranes (Biosharp, Beijing, China). The non-specific binding sites on the membrane were then blocked with 5% fresh non-fat dry milk in Tris buffered saline TBS with Tween (10 mM Tris, 150 mM NaCl, pH7.4) with 0.1% Tween 20) for 2 h. Subsequently, they were incubated with primary antibodies (anti-IL-2, anti-STAT1, anti-SHP2, anti-JAK1, and anti-p-JAK1; 1:1000 dilution; Abcam, Cambridge, UK) and primary β-actin antibody (1:1,000 dilution; abmart, Shanghai, China) overnight at 4°C, respectively. After this, the membrane was washed followed by three 10 min washes in TBST, and then incubated with anti−mouse secondary antibody (1:2000 dilution; Cell Signaling Technology, Shanghai, China) for 2 h at room temperature followed by three 10 min washes in TTBS. Finally, the chemiluminescent signals were observed with a multifunctional gel imaging system (Bio-Rad, USA) and densitometry for immunoreactive bands was performed with National Institutes of Health software (Image J).

Lung tissue was sliced and dewaxed to water (xylene I 15 min, xylene II 15 min, xylene III 15 min, absolute ethanol I 5 min, absolute ethanol II 5 min, 85% alcohol 5 min, 75% alcohol 5 min, and distilled water). After repairing with citric acid antigen repair buffer, add 3% hydrogen peroxide to incubate. After blocking at room temperature for 30 minutes and adding rabbit polyclonal anti-p-JAK1 (1 : 200 vol/vol, Abcam), anti-p-STAT1 (1 : 100 vol/vol, Abcam), and anti-SOCS3 (1 : 100 vol/vol, Abcam), put it in a wet box at 4°C refrigerator overnight. After washing with PBS, adding secondary antibody (goat anti-rabbit) (1 : 200), DAB color development, and observing under the microscope, the reaction was terminated when it showed brown. Then, hematoxylin counterstained and returned to blue, and the neutral resin sealed.

Protein Lysis Buffer (Beyotime, Shanghai, China) was used to extract the protein from cells or arterial tissues of mice. BCA kit (Nanjing Jiancheng Biogngineering Institute Nanjing, China) was used to detect the protein concentration. Then, 30 μg proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyethylenedi fluoride (PVDF). The membrane was blocked with 3% skim milk for 1 h at room temperature followed by the incubation of the primary antibodies at 4°C overnight. The primary antibodies used as follows: anti-Bax (1:1000, Abcam, Cambridge, MA, USA), anti-p-Akt 1:1000, Abcam), anti-Bcl-2 (1:1000, Abcam), anti-Cleaved caspase 3 (1:1000, Abcam), anti-p-JAK1 (1:1000, Abcam), anti-JAK1 (1:1000, Abcam), anti-p-STAT3 (1:1000, Abcam), anti-STAT3 (1:1000, Abcam), anti-β-actin (1:1000, Abcam). The β-actin worked as an internal control. Next, the membranes were incubated with goat anti-rabbit or mouse IgG secondary antibodies (1:5000, Abcam) for 1 h at room temperature. Finally, the protein bands were detected using an efficient chemiluminescence (ECL) kit (Thermo Fisher Scientific).