Ecl western blotting substrate

Protein was extracted from hippocampal tissues of the sham (n = 3), MCAO/R (n = 3), and EA (n = 3) groups using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The protein concentration was determined using the BCA Protein Assay Kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Next, an equal amount of protein (40 μg) was separated by 10% SDS-PAGE and transferred onto 0.22 μm polyvinylidene fluoride (Millipore, Bedford, MA, USA). After the blockade of nonspecific protein signals using 5% skimmed milk, the membranes were hybridized overnight with primary antibodies against Pak4 (1:1,000; ImmunoWay Biotechnology Company, Plano, TX, USA), Akt3 (1:1,000; ImmunoWay Biotechnology Company, Plano, TX, USA), Efnb2 (1:1,000; ImmunoWay Biotechnology Company, Plano, TX, USA), and Gapdh (1:10,000; ImmunoWay Biotechnology Company, Plano, TX, USA) at 4°C. On the next day, the membranes were probed for 1 h with goat–anti-rabbit or goat–anti-mouse (1:10,000; ImmunoWay Biotechnology Company, Plano, TX, USA) secondary antibodies conjugated with horseradish peroxidase at room temperature. Finally, protein bands were detected using the ECL Western Blotting Substrate (Beijing Solarbio Science & Technology Co., Beijing, China).

Total protein was isolated from LX‐2 cells using RIPA (Solarbio) and quantified using a BCA Protein Assay Kit (Solarbio). Then equal amounts of protein samples were separated on SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 60 minutes, and incubated with primary antibodies against CAPRIN1 (1:500; Proteintech), Cyclin D1 (1:1000; Bioss, Beijing, China), Cyclin E1 (1:1000; Proteintech), p‐RbS807 (1:1000; Bioss), TGF‐β2 (1:1000; Bioss), COL1A1 (1:1000; Bioss), α‐SMA (1:1000; Bioss), p‐Smad2^ser467 (1:1000; Cell Signaling Technology, Trask Lane Danvers, MA), Smad2 (1:1000; Cell Signaling Technology), p‐Smad3^S423/S425 (1:1000; Cell Signaling Technology), Smad3(1:1000; Cell Signaling Technology) and β‐actin (1:1000; Santacruz Biotechnology) at 4°C overnight. Goat anti‐rabbit IgG or goat anti‐mouse secondary antibodies (1:3000; Solarbio) were employed at 37°C for 60 minutes. The bands were visualized using electro‐chemi‐luminescence (ECL) Western Blotting Substrate (Solarbio). The protein expression was standardized to β‐actin and quantified using Gel‐Pro‐Analyzer software (Media Cybernetics, Rockville, MD).

Cells were transfected with the indicated plasmids for 4 h. 72h after the transfection, cells were collected, and lysed using Radioimmunoprecipitation assay (RIPA) buffer and protease and phosphatase inhibitors (Solarbio Science and Technology, Beijing, China). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE;Solarbio Science and Technology), then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% skim milk for 2 h and then incubated at 4°C for 12 h with the primary antibodies recognizing the following proteins: CASP6 (ABclonal Technology, Wuhan, China, Catalog NO: A19552) and α -Tubulin (Abways Biotechnology, Shanghai, China, Catalog NO: AB0049). Secondary goat anti-rabbit IgG antibodies (Abways Biotechnology, Catalog NO: AB0101) were then incubated with the membrane for 1 h at 25°C. The ECL Western Blotting Substrate (Solarbio Science and Technology, Catalog NO: PE0010) was used to visualize the immunoreactive proteins, which were detected and analyzed using the BioRad ChemiDoc imaging system (Bio-Rad, USA).