Arg54150

Tissue proteins from each sample were denatured in 5× SDS-PAGE loading buffer and electrophoresis was performed on 12% tris-glysine agarose gels. The separated proteins were transferred to nitrocellulose membrane followed by blocking with 5% non-fat milk for 2 h at room temperature. The membrane was then incubated at 4°C over-night using primary antibodies for IFNg (ARG21502, Arigo), IL1a (ARG56667, Arigo), IL6 (ARG56625, Arigo), IL12a (bs-0767R, Bioss), IL12b (bs-10641R, Bioss), IL17a (ARG55256, Arigo), TNFa (ARG10158, Arigo), IL10 (ARG21475, Arigo), TGFb (ARG10002, Arigo), CXCR4 (ARG54674, Arigo), STAT3 (ARG54150, Arigo) and Tubulin (AC015, Abclonal). This was followed by incubation with horseradish peroxidase labeled secondary antibody (AS028, AS014, Abclonal; bs-0296G-HRP, Bioss) for 2 h at room temperature. Band intensity was measured by Tanon-5200 and Tanon MP.

Radio-Immunoprecipitation Assay (RIPA) buffer was used to extract the protein in the heart tissue. The protein lysate is separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with the antibody NOX2 (A19701) and β-actin (A3718) were purchased from ABclonal, α-SMA (ARG66381) were purchased from arigo, ERK 1/2 (11257-1-AP), p-ERK 1/2 (28733-1-AP), AKT (10176-2-AP) and p-AKT (28731-1-AP) were purchased from Proteintech), The antibodies against stat3 (ARG54150) were purchased from arigo Biolaboratories. The antibodies against p-STAT3 (9145) and NFAT2 (8032) were purchased from Cell Signaling Technology, overnight at 4°C, and then incubated with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. All blots are developed and labeled using electrochemiluminescence (ECL) Plus chemiluminescence system. The intensity of protein bands was measured by ImageJ. β-actin was used as an internal control.