Odyssey infrared imaging system 3

The penumbra tissue was treated with RIPA lysis buffer (Yamei, Suzhou), and the protein concentration was measured. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the target proteins, which were then transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and then incubated with 5% BSA containing corresponding primary antibodies, including JAK2 (YT2426, Immunoway, Plano, TX, USA) (1 : 400), p-JAK2 (3776, Cell Signaling Technology, Danvers, MA, USA) (1 : 500), STAT6 (YT4454, Immunoway) (1 : 400), p-STAT6 (YP0256, Immunoway) (1 : 400), Bcl-2 (YT0470, Immunoway) (1 : 500), Bax (YT0455, Immunoway) (1 : 500), and GAPDH (YM3029, Immunoway) (1 : 5,000) on a shaker at 4°C overnight. On the second day, Tris-buffered saline-tween (TBST) was used to rinse the membranes 3 times, which were then incubated with secondary antibodies (RS23710, RS23920, Immunoway) (1 : 10,000) for 1 h. The bands were observed using an Odyssey Infrared Imaging System 3.0.29 (LICOR, Nebraska, USA).

The extracted protein of the penumbra was homogenized in RIPA lysis buffer (Yamei, Suzhou) according to the manufacturer's instructions. The supernatant of proteins was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, which were sealed with 5% skimmed milk for 2 h at room temperature. The rinsed membrane and primary antibody were incubated together overnight at 4°C, including p-JAK1 (Cat#YP0154, Immunoway, dilution 1:500), p-STAT6 (Cat#YP0256, Immunoway, dilution 1:400) and GAPDH (Cat#YM3029, Immunoway, dilution 1:5000). After 3 washes with TBST, samples were exposed for 2 h at room temperature to secondary antibodies (Immunoway, dilution 1:10,000). The bands were observed using an Odyssey Infrared Imaging System 3.0.29 (LICOR, Nebraska). All experiments were repeated three times.