Ab227503

In the immunoprecipitation experiments, whole tissue and cell lysates were prepared after I/R and incubated with the indicated antibodies together with protein A/G beads (MedChem Express) overnight. NLRX1 (1:200, Finetest, FNab05759), TRAF6 (1:200, Santa Cruz Biotechnology, sc-8409), and SHP-1 (1:200, Abcam, ab227503) antibodies were used to immunoprecipitate NLRX1, TRAF6, and SHP-1, respectively. The beads were then washed 4 times with lysis buffer, and the immunoprecipitates were separated by SDS-PAGE. The proteins were transferred to PVDF membranes (Millipore, Boston, MA, USA) and then incubated with the indicated antibodies.

Total protein from cultured astrocytes and ischemic brain tissues was prepared using RIPA buffer with the protease inhibitor PMSF. A total of 50 μg of protein (per lane) was separated by SDS-PAGE (different percentages of SDS-PAGE gels were used to separate and analyze the different proteins) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membranes were then blocked with 5% nonfat milk in TBST buffer for 2.0 h at 37 °C and incubated with the following primary antibodies overnight at 4 °C: DJ-1 (1:1000, Abcam, ab76008), anti-β-actin (1:3000, Proteintech, 66009-1-Ig), IL-1β (1:200, Boster, PB0055), IL-6 (1:200, Boster, PB0061), TNF-α (1:200, Boster, PB0082), NLRX1 (1:200, Finetest, FNab05759), TRAF6 (1:200, Santa Cruz Biotechnology, sc-8409), and SHP-1 (1:200, Abcam, ab227503). The next day, the membranes were incubated with secondary antibodies at 37 °C for 2 h. The densities of the bands were detected using an imaging densitometer (Bio-Rad), and the gray values of the bands were quantified using ImageJ. Relative protein expression was normalized to β-actin staining intensity.

Total protein was extracted with RIPA lysate containing cocktail protease inhibitor (Beyotime, China), and nuclear protein was extracted by the CelLytic NuClear Extraction Kit (Sigma, USA). The protein concentration was detected with the BCA Protein Assay Kit (Beyotime, China). Protein lysates were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Then, PVDF membranes were blocked with 5% nonfat milk and incubated with primary antibodies. The following primary antibodies were used in this study: anti-METTL14 (ab252562, Abcam), anti-PTPN6 (ab227503, Abcam), anti-RUNX2 (ab76956, Abcam), anti-PPARγ (ab59256, Abcam), anti-C/EBPα (ab40764, Abcam), anti-C/EBPβ (ab15049, Abcam), anti-β-catenin (ab6302, Abcam), anti-p-β-catenin (ab11350, Abcam), anti-GSK-3β (ab32391, Abcam), anti-p-GSK-3β (ab68476, Abcam), anti-GAPDH (ab8245, Abcam), anti-Histone H3 (ab1791, Abcam). GAPDH and H3 were used as the internal controls for total protein and nuclear protein, respectively. Horseradish peroxidase-conjugated secondary antibody was used to develop the blots. Blots were detected with the ECL Western Blotting Substrate Kit (ab65623, Abcam) on an ECL system (Bio-Rad, USA).