Anti nrf2

On day 3 post-MCAO, ipsilateral hemispheres were homogenized in RIPA lysis buffer (Sigma, USA) and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF) (Sigma, USA). After centrifugation, the supernatants were collected as total proteins. Proteins were loaded and transferred to a PVDF membrane (Millipore, USA). After being blocked, membranes were incubated overnight at 4 °C with anti-Nrf2 (1:1000, Millipore), anti-HO-1 (1:1000, Millipore), or rabbit polyclonal antibodies. Membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:4000, Vector, Burlingame, USA). The membranes were placed into a gel imaging system (Bio-Rad, ChemiDoc XRS, USA) and then exposed. The intensity of blots was quantified using the Quantity One Analysis software (Bio-Rad, USA). β-Actin was used as an internal control.

Protein analysis and immunoblotting were performed as previously described [13 (link), 14 (link)]. The following primary antibodies were used: anti-β-ACTIN Sigma-Aldrich (Castle Hill, NSW, AUS), anti-NRF2 (Merck KGaA, Darmstadt, Germany), and antibodies against phospho-histone H2A.X, cleaved PARP and HMOX1 (Cell Signaling Technology, Inc., MA, USA).

HEK293 cells were co-transfected with pcDNA3.1-FLAG Nrf2 [44 (link)] construct, together with pJTI-R4-DEST-CMV-N-EmGFP empty vector (Life Technology, Carlsbad, CA), pJTI-R4-DEST-CMV-N-EmGFP BRD3 (Addgene, Cambridge, MA), pcDNA4-HA empty vector or pcDNA4-To-HA-BRD4FL (Addgene) construct, using Lipofectamine LTX (Life Technology) for 48 hours, followed by protein extraction. Protein extracts from 1500μg of cell lysate were immunoprecipitated with 5μg of anti-BRD3 or anti-BRD4 antibody (Bethyl Laboratories) overnight at 4°C. Immunoprecipitates were captured by Protein G-Sepharose (GE Healthcare, Buckinghamshire, England), and then released by boiling in sample buffer, followed by immunoblot analysis with anti-BRD3, anti-BRD4 and anti-Nrf2 (Merck Millipore, Billerica, MA) antibodies.

Under deep anesthesia, the rats were perfused with paraformaldehyde and the brains were harvested. Brain tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Coronal sections (4 μm) were treated with xylene and gradient alcohol, then blocked with 5% bovine serum albumin for 45 min at room temperature. The sections were incubated with the following primary antibodies, including anti-GPX4 (1:200, cat.no. A1933, Abclonal; anti-NeuN (1:200, cat.no. MAB377, Merck Millipore); anti-Nrf2 (1:100, cat.no. A11159, Abclonal) overnight at 4°C. Subsequently, after being rinsed three times with PBST, the sections were dried, and the fluorescent secondary antibody was added dropwise for incubation at 37°C for 1 h in a wet box, then 4,6-diamidino-2-phenylindole (DAPI) was added for incubation at room temperature for 15 min. Finally, we sealed the slides using a mounting liquid that contains the anti-fluorescence quencher. The right temporal cerebral cortex images were observed, photographed and collected under the fluorescence microscope.