Ecl detection kit

The protein was extracted by RIPA lysis buffer supplemented with protease inhibitor (1:50) and PMSF (1:100) (ZHHC Biotechnology, Xi’an, China). The supernatant was collected by centrifugation and the protein concentration was determined by the BCA method (Beyotime, Shanghai, China). A total of 30 ug of protein added to each lane was separated by SDS-PAGE gel and then transferred to an NC membrane. After blocking with 5% milk, the NC membrane was incubated with the primary antibody overnight at 4 °C. Rabbit anti-NRF2 (1:3000, Proteintech, 16396-1-AP, Wuhan, China), Rabbit anti-P62 (1:3000, Proteintech, 18420-1-AP, Wuhan, China), Rabbit anti-BECLIN1 (1:1000, Abcam, ab207612, Cambridge, UK), Rabbit anti-β-Actin (1:1000, Proteintech, 20536-1-AP, Wuhan, China), Rabbit anti-BCL-2 (1:1000, Proteintech, 12789-1-AP, Wuhan, China), Rabbit anti-BAX (1:3000, Proteintech, 50599-2-Ig, Wuhan, China) and Rabbit anti-LC3 (1:1000, Proteintech, 14600-1-AP, Wuhan, China) were used. After incubation with the peroxidase conjugated secondary antibody for 1 h at RT, the protein bands were detected with an ECL detection kit (YEASEN, Shanghai, China).

Western immunoblot analysis was used to assess protein expression of HIF-1α, VEGF, VEGFR2, and Notch1. Three rats of each group were sacrificed after anesthesia. Right brains tissues were removed immediately and stored at −80°C until analysis. The proteins were isolated from the ischemic cortex tissue after lysis in RIPA buffer (Beyotime, Shanghai, China), and protein determination was quantitated using a BCA Protein Assay kit (Beyotime, Shanghai, China). Denatured protein samples were separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with primary antibodies: rabbit polyclonal anti-HIF-1α (1 : 500, BIOSS) and rabbit polyclonal antibody anti-VEGF (1 : 500, BIOSS), overnight at 4°C. Then, after rinsing in TBST buffer for 5 min three times, the membrane was incubated with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1 : 10000, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h. Finally, the protein bands were visualized using enhanced chemiluminescence (ECL) detection kit (Yeasen Biotech, Shanghai, China) and were quantitatively analyzed by using ImageJ software (Rawak Software Inc., Germany).

Proteins were extracted using a radioimmunoprecipitation assay (Boster, China), and protein content was estimated using the bicinchoninic acid assay (Boster). They were electrophoresed in 10% sulfate-polyacrylamide gel. Subsequently, the proteins were transferred onto polyvinylidene fluoride membranes, washed with Tris-buffered saline with Tween 20, and blocked with 5% BSA blocking buffer at 37°C for 2 h. Then, primary antibodies, AFF4 (ab103586, Abcam, UK) and GAPDH (ab8245, Abcam), were incubated with the membranes overnight at 4°C, prior to incubation with secondary antibody (ab6721, Abcam) for 1 h at 37°C. The ECL detection kit (Yeasen, China) and ImageJ software (Version 1.48; NIH, USA) were used to visualize and determine the protein levels [27 (link)].