Ab28815

The expression levels of STAT1, STAT4, STAT6 and the corresponding phosphorylated proteins in lung digests were analyzed by Western blot analysis. Cell lysates (40 μg) were separated by 10% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, USA). After incubation in a blocking buffer containing 5% skim milk in TBST (12.5 mM Tris–HCl pH 7.5, 68.5 mM NaCl, and 0.1% Tween 20) for 1 h, the blots were incubated overnight with primary antibodies including rabbit anti-STAT1 (D1K9Y, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT1 (Tyr701, Cell Signaling Technology, USA), rabbit anti-STAT4 (C46B10, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT4 (ab28815, Abcam, UK), rabbit anti-STAT6 (ab32520, Abcam, UK), and rabbit anti-phosphorylated STAT6 (ab28829, Abcam, UK). An anti-mouse GAPDH antibody was used as a loading control. The blots were washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (Jackson ImmunoResearch) and then developed with an enhanced chemiluminescence (ECL) substrate solution (Millipore).

The paraffin-embedded rat aortic tissues were sliced and the sections were routinely dehydrated with gradient ethanol and next immersed in potassium citrate at 90 °C for 10 min followed by antigen retrieval. The sections were washed with PBS three times and next added with 3% H₂O₂ to inactivate endogenous peroxidase for 10 min. After blocked with goat serum (Solarbio, Beijing, China) for 20 min, the sections were incubated with anti-rabbit Cbl (1: 200, PA5-8292, Invitrogen, Carlsbad, CA, USA), anti-rabbit p-JAK2 (1: 2000, ab32101, Abcam, UK), anti-rabbit p-STAT4 (1: 100, ab28815, Abcam), and anti-mouse Runx3 (1: 500, ab135248, Abcam) overnight at 4 °C. The samples were then incubated with secondary goat anti-mouse immunoglobulin G (IgG) (ab150113, Abcam) or goat anti-rabbit IgG (ab150077, Abcam) for 1 h at room temperature. Finally, the sections were developed with diaminobenzidine (DAB; ZLI-9017, ZSGB-BIO, Beijing, China) and 4 sections of each sample were observed under microscope (DMI3000, Leica Biosystems, Shanghai, China) in three random fields, as positive cell percentage was calculated.