Ecl chemiluminescence substrate kit

Retinal tissues were lysed with 100 μL of RIPA tissue lysates, and the supernatant was discarded after centrifugation (15,000 rpm) at 4°C for 15 min. Then, the protein concentration and purification were determined by BCA method. The protein extracts were separated with agarose gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were placed in solution containing 5% fat-free milk powder and blocked with gentle shaking at room temperature for 1 h. Afterwards, these membranes were incubated with primary polyclonal antibodies against p-PI3K(1:1,000, rabbit, Bioss, bs-20611R) and p-AKT (1:1,000, rabbit, Bioss, bs-2720R) and β-actin monoclonal antibodies (1:5,000, mouse, Proteintech, 66009-1-Ig) overnight at 4°C. After washing three times, secondary antibodies (IgG-HRP, 1:5,000, goat anti-rabbit, Abbkine, A21031; IgG-HRP, 1:5,000, goat anti-mouse, Abbkine, A21010) were added to incubate for 1 h. After washing, the protein bands were developed with ECL chemiluminescence substrate kit (BL520B, Biosharp). β-actin was used as a loading control. Finally, densitometry analysis was performed using ImageJ software, and the relative expression levels were calculated by using the gray value of the target strip than the gray value of β -actin.

Referring to the previous description (32 (link)), protein extraction and immunoblotting were performed. The antibodies directed against the following proteins, including PI3K, p-PI3K, AKT, p-AKT, and β-actin were used, and their directions are shown in Table 1. HRP-conjugated secondary antibodies (1:5,000, GeneTex, Irvine, CA, USA) were also used. Other materials consisted of electrophoresis and membrane transfer device (Bio-Rad), RIPA lysate (Beyotime), protease inhibitor (cocktail) (Roche), ECL chemiluminescence substrate Kit (Biosharp, Hefei, China), defatted milk powder (Wondersun, Harbin, China), protein marker (Bio-Rad), BCA kit (Beyotime), and SDS-PAGE gel rapid preparation kit (Beyotime). Blotting was captured by Molecular Imager ChemiDocTM XSR+ Gel Imaging System (Bio-Rad) and analyzed using ImageJ software (NIH). In semiquantitative analysis of the target protein, each sample was normalized to β-actin.

Proteins were extracted from kidney tissues using RIPA lysis buffer and centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was extracted for use. The protein concentration was determined by a BCA kit (Beyotime). After separation using 12.5% SDS-PAGE gels (EpiZyme), proteins were transferred to PVDF membranes and the membranes were sealed in 5% skim milk for 1 h. Then, the membranes were incubated with primary antibody 3²-actin (1:5000, mouse antibody, 43 KD, Affinity), MMP9 (1:1000, rabbit antibody, 78 KD, Affinity) in a vertical shaker at 4°C for 16 h. After rinsing, sheep anti-rabbit IgG or sheep anti-mouse IgG (1: 5,000, Abbkine) was added and incubated for 1 h at room temperature. Finally, an ECL chemiluminescence substrate kit (Biosharp) was used for development, image information was acquired in the gel imager, and the data were processed using ImageJ software.