Rs23920

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).

Total protein was extracted from the cells using 1 × RIPA lysis buffer (P10013B, Beyotime, Shanghai, China). The concentration of protein was measured by using BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China). And then equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membrane (NC membrane). After electroblotting, the membranes were blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated with primary antibodies for LDHA (1:2000, Santa Cruz sc-137243, Texas, USA), PFKM (1:1000, ABclonal A5477, Shanghai, China), NAT10 (1:1000, ABclonal A19286, Wuhan, China), YTHDC1 (1:500, Proteintech 14,392–1-AP, Wuhan, China), ac4C (1:1000, Abcam ab253039, Cambridge, UK) or β-actin (1:5000, Abcam ab253039, Cambridge, UK). The membrane was then incubated with anti-mouse secondary antibodies (RS23910, ImmunoWay, USA) or anti-rabbit secondary antibodies (RS23920, ImmunoWay, USA). Results were detected using Odessey Clex (LI-COR, USA), followed by further analysis.

The total proteins were extracted from cells or tissues using 1 × RIPA lysis buffer supplemented with a protease inhibitor cocktail (Roche). The concentrations of extracted total protein samples were measured by a BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes. After blocking the membranes with non-fat milk (5% w/v) for 1 h, the proteins were incubated at 4 °C overnight with the primary antibodies against CD63 (1:1,000, WL02549, Wanleibio, China), CD81 (1:1,500, GTX101768, Gene Tex, USA), TSG101 (1:1,500, GTX70255, Gene Tex), ACSL4 (1:10,000, ab155282, Abcam, UK), GPX4 (1:1,000, A13309, Abclonal, USA), GAPDH (1:5,000, AC002, Abclonal), or β-Tubulin (1:5,000, AC021, Abclonal). Next, the membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (RS23910 and RS23920, ImmunoWay, USA) in dark at room temperature for 1 h. The membranes were scanned, and the gray values of protein band were detected by Odessey CLx (LI-COR, USA).