Rabbit anti gapdh

Total cellular proteins (20 μg from each sample) obtained above were separated by SDS-PAGE (Solarbio, Beijing, China) under reducing conditions and transferred onto a nitrocellulose membrane (Invitrogen, Shanghai, China). Membranes were blocked in 5% dry milk and probed overnight at 4 °C with gentle rocking with anti-Runx-2 (1:1000, Abcam, Cambridge, UK), rabbit anti-BMP-2 (1:1000, Abcam), rabbit anti-COL-1 (1:1000, Abcam), mouse anti-acetylated α-tubulin (1:1000, Abcam), rabbit anti-Dynlt1 (1:800, Abcam), rabbit anti-IFT88 (1:500, Santa Cruz Biotech, Dallas, Texas), or rabbit anti-GAPDH (1:800, Bioworld, Shanghai, China). After washes, membranes were incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG, 1:10000, Bioworld). The proteins recognized by the antibody complexes were visualized by chemiluminescence (Solarbio, Beijing, China). The optical density of each maker band was measured using Image-Pro Plus 6.0 software.

Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (BestBio Cat No. BB-3101) was used to extract proteins. The protein concentration was assessed using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher). Western blotting analysis was performed by loading 15 µg of lysate onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferring the gels to polyvinylidene difluoride (PVDF) membranes (Millipore), and incubated with rabbit anti-GDF5 (1:1000, #A13167; ABclonal), rabbit anti-PPARγ (1:1000, #2443; CST), rabbit anti-FASN (1:1000, #D262701; Sangon Biotech), rabbit anti-C/EBPα (1:1000, #2295; CST), rabbit anti-FABP4 (1:1000, #2120; CST), rabbit anti-CD36 (1:1000, #ab1336-25; Abcam), rabbit anti-GAPDH (1:5000, #BS65529; Bioworld), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381538; ZEN BIO), rabbit anti-Alix (1:1000, #D262028; Sangon Biotech) or rabbit anti-Calnexin (1:1000, #D262986; Sangon Biotech). Afterward, goat anti-rabbit secondary antibody (1:50000, # BS13278, Bioworld) conjugated with HRP was used. GAPDH levels served as the loading control. The amount of protein was measured using ImageJ software.

For direct detection of protein levels, the heart was quickly cut off after 120 min of reperfusion; total protein was obtained from the heart tissue according to the manufacturer's instructions. Protein concentration was determined by BCA Protein Assay Kit (KeyGen BioTECH, China). 30 ug of total proteins was separated by electrophoresis on SDS-PAGE and then transferred onto 0.45 μm PVDF membranes. The membranes were blocked for 2 h in 5% skim milk at room temperature and incubated with primary antibodies at 4°C overnight. Immunoblots were performed using the following antibodies: RAGE (Cell Signaling Technology, USA), HMGB1 (Cell Signaling Technology, USA), phosphorylated ERK1/2 (Cell Signaling Technology, USA), total ERK (Jiancheng Technology, CHINA), phosphorylated Akt (Cell Signaling Technology, USA), total Akt (Jiancheng Technology, CHINA), and rabbit anti-GAPDH (Bioworld, China). Then the membranes were washed with TBST. We blocked the protein with phosphate-horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA) at room temperature for 2 hours. The complexes were detected by using ECL kit (Thermo, USA). The images were analyzed with digital gel imaging system (Bio-Rad, USA).

The 6 raw herbs of DSS were purchased from Kangmei Pharmaceutical Limited Company (Guangzhou, China). Donepezil hydrochloride (D849374) and scopolamine hydrobromide trihydrate (S860151) were from Macklin (Shanghai, China); FITC-dextran (^#46944) was from Sigma system (ChemiDoc MP, Bio-Rad, California, USA); total cholesterol Assay Kit (A111-1-1) and Triglyceride Assay Kit (A110-1-1) were from Nanjing Jiancheng; and Malondialdehyde (MDA) ELISA Kit (JL13339), Adiponectin (ADPN) Kit (JL20696D), Low-Density Lipoprotein Cholesterol (LDL-C) Kit (JL20313), High-Density Lipoprotein Cholesterol (HDL-C) Kit (JL20356), Tumour Necrosis Factor-α Kit (TNF-α) (JL10484) and Interleukin 6 (IL-6) Kit (JL20313) were from Jiang Lai Biological (Shanghai, China). Antibodies included rabbit anti-ZO-1 (PB9234, Boster, Pleasanton, CA, USA), rabbit anti-OCLN (A01246-2, Boster), rabbit anti-OCLN (#91131, CST, Danvers, MA, USA) in brain immunofluorescence, rabbit anti-ZO-1 (#13663, CST) in brain immunofluorescence, rabbit anti-PPAR-γ (^#2435, CST), Cy3-labelled Goat Anti-Rabbit IgG (H+L) (A0516, Beyotime, Shanghai, China), rabbit anti-LXR alpha+beta (ab21669, Abcam, Cambridge, MA, USA), rabbit anti-actin (^#2118, CST), and rabbit anti-GAPDH (AP0063, Bioworld, St. Louis Park, MN, USA).