Glyceraldehyde 3 phosphate dehydrogenase gapdh

Protein in the ischemic cortex tissue was extracted with a commercial protein extraction kit (Beyotime), and the protein quantity was measured with a BCA protein assay kit (Beyotime), following the manufacturer's protocols.
A total of 40 μg protein samples were separated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (10–15%), and the protein was transferred to nitrocellulose membranes. After the samples were blocked in 10% skimmed milk for 1 h at room temperature, they were incubated with primary antibodies against LC3 (1 : 1000; Sigma-Aldrich), p62 (1 : 1000; Abcam), p-AMPK (1 : 1000; Cell Signaling Technology, MA, USA), AMPK (1 : 1000; CST), Sirt1 (1 : 1000; CST), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 3,000; Bioworld Technology, St Louis Park, USA) at 4°C overnight. The membranes were washed with PBS 3 times, for 5 min each time, and incubated with horseradish peroxidase-conjugated IgG (1 : 5,000, Cell Signaling Technology) secondary antibodies for 2 h at room temperature. Specific protein signals were visualized with an ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). The bands were analyzed by ImageJ software and normalized for GAPDH expression.

First, equal amounts of protein were leaded in each lane of a polyacrylamide gel. Next, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C. The membrane was then washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated IgG. Finally, we employed ECL Western blotting detection reagents (Thermo Fisher Scientific) to visualize the protein bands and used Un-Scan-It 6.1 software (Silk Scientific, Orem, UT, USA) to measure the band density.

The cells were lysed in RIPA buffer, and the proteins (20 μg) were separated on 10% SDS/PAGE gels and then transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking the membranes with 5% non-fat milk, they were incubated with the appropriate dilutions of specific primary antibodies (1:1000). The blots were then incubated with HRP-conjugated secondary antibodies (1:4000). Antibodies against TRIM36 (Sigma, GER), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Bioworld Technology, USA), extracellular signal-regulated kinase (ERK), p-ERK, p-MSK1, c-myc, cyclin D1, and cyclin E1 (Cell Signaling Technology, USA) were used in Western blot analysis in accordance with the manufacturer’s instructions. The blots were detected using enhanced chemiluminescence (Thermo Scientific). The protein levels were determined by normalization to GAPDH.