Bs6007m

Total protein was extracted from cultured neurons and the ischemic penumbra of the rat cortex using cell lysis buffer supplemented with proteinase and phosphatase inhibitors. The nuclear proteins were extracted using a commercial kit (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked in 5% non-fat milk TBST buffer for 1.5 h at room temperature. The membranes were incubated in primary antibody overnight at 4 °C and in secondary antibody for 1 h at room temperature. Dilutions for primary antibodies were as follows: anti-GSK-3β (#9315, 1:1000, Cell Signaling Technology, Boston, MA, USA), anti-β-catenin (#9582, 1:1000, Cell Signaling Technology), anti-Nrf2 (YT3189, 1:500, Immunoway, Houston, TX, USA), anti-GSK-3β (phospho-tyr216) (ab75745, 1:500, Abcam, Cambridge, MA, USA), anti-LaminB1 (ab133741, 1:500, Abcam), anti-HO-1 (BS6626, 1:500, Bioworld, St. Louis Park, Minnesota, USA), anti-NQO1 (BS6833, 1:500, Bioworld), and anti-actin (BS 6007 M, 1:10,000, Bioworld).The secondary antibody was diluted 1:5000 (Sangon Biotech, S hanghai, Co., Ltd.). The density of bands was detected using an imaging densitometer (Bio-Rad, Foster City, CA, USA), and the gray value of bands was quantified using Quantity One 1-D analysis software.

Tissue and cell proteins were separated by 10% SDS/PAGE gels and then transferred to nitrocellulose membranes (BioTrace™ NT, New York City, NY, United States). After blocking with 5% skim milk, the membrane was incubated with primary antibodies at 4°C overnight. The antibodies are listed as follows: total-AMPK (Abcam, ab80039, 1:2000, Cambridge, United Kingdom), phospho-AMPK at Thr172 (Abcam, ab23875, 1:2000, Cambridge, United Kingdom), total-CREB (Abcam, ab32515, 1:2000, Cambridge, United Kingdom), phospho-CREB at Thr133 (Abcam, ab32096, 1:2000, Cambridge, United Kingdom), total-GSK3β (Proteintech, 22104-1-AP, 1:1000, Wuhan, China), phospho-GSK3β at Ser389 (Proteintech, 14850-1-AP, 1:1000, Wuhan, China), PEPCK (Proteintech, 16754-1-AP, 1:1000, Wuhan, China), G6PASE (Novus, NBP1-80533, 1:1000, Littleton, CO, United States), and β-actin (Bioworld, BS6007M, 1:4000, Minneapolis, MN, United States). Then, HRP-conjugated secondary antibody (Sungene Biotech, LK 2001, 1:5000, Tianjin, China) was incubated for 1 h at room temperature. Finally, the protein bands were developed by an ECL kit (Advansta, Menlo Park, CA, United States). The quantification of protein bands was analysed by ImageJ software.

Proteins were extracted from cells or tissues in RIPA lysis buffer supplemented with PMSF and phosphatase inhibitors and dissolved in SDS loading buffer. Equal amounts of proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Millipore). After being blocked with 5% skimmed milk in 0.25% TBS-Tween (TBST), membranes were incubated with the following primary antibodies overnight at 4°C: p62 (1:2000, Abcam, ab56416), ATG5 (1:2000, Proteintech, No. 10181-2-AP), Beclin-1 (1:2000, Proteintech, No. 11306-1-AP), LC3 (1:2000, Proteintech, No. 14600-1-AP), TET2 (1:1,000, Proteintech, No. 21207-1-AP), p-AMPK (1:2000, Abcam, ab23875), AMPK (1:2000, Abcam, ab80039), SGLT2 (1:1,000, Proteintech, No. 24654-1-AP), SREBP-1c (1:1,000, Abcam, ab28481), PPARα (1:800, Proteintech, No. 15540-1-AP), CD36 (1:1,000, Abcam, No. 18836-1-AP), and β-actin (1:5,000, Bioworld, BS6007M). After washing with TBST, the horseradish peroxidase (HRP)-conjugated secondary antibody was incubated for 1 h at room temperature. Finally, protein bands were visualized with an ECL kit (Advansta, K-12045-D50). Quantification of each band was analyzed with ImageJ software. Proteins levels were normalized against the loading control β-actin.

Cells or tissue samples were lysed in RIPA lysis buffer (Tian P. et al., 2017 (link)) added protease inhibitor (P8340, Sigma, St. Louis, MO, United States) for 30 min on ice, and then centrifuged at 12, 000 rpm for 15 min at 4°C. The supernatant was collected and measured to calculate protein concentration using a BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, PA, United States). After denaturation, 80 μg protein was electrophoresed in a 10% SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (Tris buffer with 0.1% Tween, pH 7.6) for 2 h and then incubated at 4°C overnight with primary antibodies: DMT1 (ab55735, Abcam, 1:500) or β-actin (BS6007M, Bioworld, 1:10000). A secondary HRP-conjugated antibody (BS12478, Bioworld, 1:10000) was used to incubate the membrane for 2 h prior to chemiluminescent detection. The signals were determined using a chemiluminescent substrate (ECL) kit (NCI4106, Thermo Fisher Scientific, Waltham, PA, United States). Then, protein intensities were quantified by the VersaDoc MP 4000 system (Bio-Rad, California, CA, United States).