Anti phosphorylated stat1

Proteins were extracted from transfected Hs294T cells and separated by SDS-PAGE, transferred to a NC membrane (GE Healthcare), blocked and probed with primary specific antibodies, followed by the appropriate secondary antibodies. After washing, the immunoreactive complexes were detected using a chemilumenescence reagent (Biodragon, Beijing, China). Antibodies used in this experiment were as follows: anti-phosphorylated STAT1 (Cell Signaling Technology), anti-CDH5 (Cell Signaling Technology), anti-TFPI2 (Abcam, Cambridge, MA, USA), anti-β-actin (Bioworld Technology, St.Louis, MN, USA).

RIPA buffer was added into M1‐type polarized macrophages, which were incubated on ice for 30 minutes. During incubation, samples were shaken every 10 minutes, and then centrifuged at 12 000 rpm for 15 minutes at 4°C. SDS loading buffer was added into the supernatant before being denatured at 100°C for 10 minutes, which was then followed by SDS‐PAGE. After transferring the proteins to the PVDF membranes, the membranes were blocked with 5% skim milk for two hours at room temperature. The corresponding primary antibodies (anti‐Grx1, anti‐STAT1, antiphosphorylated STAT1, antiactin or anti‐HMGN2 [Cell Signaling Technology, USA]) were then diluted, and the membranes incubated with primary antibodies at 4°C overnight. After washing the membranes three times with PBST, the secondary antibodies corresponding to each antibody were diluted 1:1000 and incubated at room temperature for two hours. The membranes were then washed three times with PBST before imaging.