Western Blot Analysis of Post-MCAO Brain

Brain samples were collected 24 h after MCAO. Western blotting was performed as described previously [35 (link)]. The proteins of right hemispheres were extracted by cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein (50 μg) were loaded and subjected to electrophoresis on an SDS-PAGE gel. After being transferred to a nitrocellulose membrane, the membranes were cut into different strips which covered an area with target protein according to molecular weight marker location and were blocked with 5% nonfat milk (Bio-Rad Laboratories, Irvine, CA, USA). The membrane was incubated with the primary antibody overnight at 4 °C. Primary antibodies included the following: anti-TGR5 (1:1000; ab72608; Abcam, Cambridge, MA,USA), anti-Pellino3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase-8 (1:1000; ab25901; Abcam, Cambridge, MA,USA), anti-NLRP3 (1:500; NBP2-12446; NOVUS, CO, USA), anti-caspase-1 (1:1000; NBP1-45433; NOVUS, CO, USA), and IL-1β (1:1000; ab2105; Abcam, Cambridge, MA, USA). β-actin (1:1000, sc-58673, Santa Cruz, TX, USA) was used as an internal loading control. The secondary antibodies were all from Santa Cruz Biotechnology. Blot bands were quantified by densitometry using ImageJ software (ImageJ 1.4, NIH, USA).