Western blotting was performed as previously described [5 (link)]. In brief, rats were anesthetized with isoflurane at the established time points after ICH and transcardially perfused with cold PBS. The right brain hemisphere was homogenized in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology, USA) and further centrifuged at 14000 rpm for 30 min. Equal amounts of protein (4 μL, 30 μg) were loaded onto a 10% SDS-PAGE gel. After separation, the proteins were transferred to a nitrocellulose membrane, which was further blocked for 2 h with a blocking solution. The membranes were incubated with the following primary antibodies overnight at 4°C: anti-albumin (1 : 500, ab207327, Abcam, USA), anti-p-ERK (1 : 1000, Santa Cruz Biotechnology, USA), anti-ERK (1 : 1000, sc-514302, Santa Cruz Biotechnology, USA), anti-Nrf2 (1 : 1000, GTX103322, Gene Tex), anti–HO-1 (1 : 1000, ab68477, Abcam, MA, USA), anti-Romo1 (1 : 1000, Aviva Systems Biology, San Diego, CA), anti-Bcl2 (1 : 1000, ab59348, Abcam, MA, USA), and anti-Bax (1 : 1000, Littleton, CO). The membranes were incubated with an anti-β-actin antibody (1 : 5000, sc-47778, Santa Cruz Biotechnology, TX, USA).) The results were normalized using β-actin as a loading control. The relative density of the blot bands was quantified by densitometry using the ImageJ software.
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