Bradford protein quantification kit

Whole cell extracts were prepared in the RIPA lysis buffer (P0013, Beyotime) and then centrifuged (14,000 rpm, 4 °C) for 15 min. The supernatants were obtained and protein concentrations were tested with Bradford Protein Quantification Kit (500-0205, Bio-Rad Laboratories). The protein samples were loaded and separated by 8–12% SDS-PAGE, then transferred to PVDF membrane (Millipore). Tris-buffered saline with 0.1% Tween containing 5% non-fat milk was used to block the membranes for 2 h at room temperature. Then the membranes were incubated with the primary antibodies targeting BNIP3 (ab10433, Abcam, 1:1000), LC3B (L7543, Sigma, 1:5000), Atg5 (12994, Cell Signaling Technology, 1:1000), ERK1/2 (4695, Cell Signaling Technology, 1:1000), phosphorylated ERK1/2 (p-ERK1/2; 4370, Cell Signaling Technology, 1:1000), P62 (88588, Cell Signaling Technology, 1:1000) and β-actin (ACTB, HRP-66009, Proteintech, 1:2000) overnight at 4 °C. Then the corresponding secondary HRP–conjugated antibodies (1:5000) were incubated for 2 h at room temperature. The blots were visualized with the ChemiDoc XRS System (Bio-Rad Laboratories) using the enhanced chemiluminescence reagent (K-12045-D10, Advansta).

The dissected brain tissues from the areas of infarction were placed in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). The protein concentration was determined using a Bradford protein quantification kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The protein-loading quantity was 40 µg on an 8-15% SDS-PAGE gel. The protein bands were separated by electrophoresis and were electroporated onto a PVDF membrane. Following blocking with 5% skim milk in a Tris-buffered saline solution (with 1% Tween-20), the primary antibody was added and allowed to incubate overnight at 4°C. The antibodies used were monoclonal rabbit anti-HMGB1 (1:1,000; cat. no. ab79823; Abcam, Cambridge, MA, USA) and monoclonal rabbit anti-RAGE (1:500; cat. no. ab3611; Abcam). The membranes were washed three times with PBS-Tween 20 and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:500; cat. no. ab7090; Abcam) for 1 h at room temperature. Development was performed using the SuperSignal™ West Pico chemiluminescent substrate (Invitrogen; Thermo Fisher Scientific, Inc.). The internal reference used was β-actin (1:500; cat. no. ab8227; Abcam), and optical-density analysis was performed using ImageJ version 1.44 (National Institutes of Health).