Fnab05759

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.