Gtx103322

The cells were lysed using RIPA lysis buffer, and the lysates were collected for Western blotting assay. In brief, equal loading volumes of cell lysates were analyzed by SDS-PAGE, followed by transfer to a PVDF membrane. The membrane samples were probed with antibodies specific for anti-DENV NS2B antibody (1:3000; GeneTex, GTX124246, Inc, Irvine, CA, USA), anti-GAPDH antibody (1:10,000; GeneTex, GTX112827), anti-Nrf2 antibody (1:3000; GeneTex, GTX103322), anti-Lamin B1 (GTX103292), anti-Tubulin (GTX112141), anti-Histone H1 (GTX87506) antibody (1:10,000; GeneTex), and anti-HO-1 antibody (1:3000; Abcam ab13243, Cambridge, MA, USA). The protein abundance of the samples was quantified using ImageJ software following densitometric scanning [20 (link)].

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.

Deparaffinized 5-μm-thick tissue slices were incubated with 3% H₂O₂ for 30 min, after which they were incubated for 1 h at 37 °C with anti-Nrf2 (GTX103322, Genetex, Alton Pkwy Irvine, CA, USA, 1:100) and anti-SIRT1 (ab110304, Abcam, Waltham, MA, USA, 1:70) reagents, following the manufacturer’s instructions. Cross-sections were treated with the secondary antibody HRP Envision kit (DAKO) for 20 min after being rinsed with PBS. The slices were then washed with PBS and given a 10-min incubation with diaminobenzidine (DAB). They were then dehydrated, cleaned in xylene, counterstained with hematoxylin, and cover slipped for microscopic analysis. The analysis was completed using the technique adopted from Elsayed et al. [58 (link)]. Seven representative nonoverlapping fields were randomly selected and scanned in order to determine the relative mean Area (%) of positive immunohistochemistry expression levels of Nrf2 and SIRT1 in immune-stained sections for each tissue section per sample. Data were gathered for the investigation of tissue sections utilizing a Full HD microscopic imaging system (Leica Microsystems Ltd.; Wetzlar, Germany) run using Leica Application software version 3.7.5 for tissue section analysis.