Enhanced chemiluminescence kit

Proteins were obtained from heart tissues after the whole perfusion process. Quantification of the protein was conducted using a modified bicinchoninic acid (BCA) assay (Cwbio, China). Protein samples were prepared by homogenizing rat heart, then being boiled for 5 minutes with loading buffer (Cwbio, China). Samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gel and transferred onto polyvinylidene fluoride (PVDF) membrane by electroblotting. After being blocked using skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the primary antibody. The following day, the membranes were washed and incubated with the goat anti-rabbit horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Cwbio, China) under a ChemiDoc imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).

After being treated with sevoflurane for 48 h, cells were harvested and lysed with RIPA Lysis Buffer (CWBIO, Beijing, China) to extract protein. Then, 20 µg protein of each sample was electrophoresed on 10% SDS-PAGE gel, and transferred onto polyvinylidene fluoride membrane (PVDF; Millipore, Billerica, MA, USA). Subsequently, the members were blocked with 5% dried skimmed milk for 1 h followed by incubation with primary antibodies (dilution, 1:1000; Proteintech Group, IL, USA) at 4 °C overnight. After labelling with horseradish peroxidase (HRP)-conjugated secondary antibodies (dilution, 1:3000; Proteintech Group) for 1 h, the signals were visualized using an Enhanced Chemiluminescence kit (CWBIO). β-tubulin was used as endogenous control, and the relative expression of proteins were normalized to NC.

Acridine orange (AO) was from Solarbio. N-Acetyl-L-cysteine (NAC) was from Sigma. Bicinchoninic acid (BCA) protein assay kit and enhanced chemiluminescence kit were from Cwbio. RIPA buffer and LysoTracker Deep Red were from Beyotime Biotechnology. For these compounds, catalog information is listed in Table 1. Western blotting and immunofluorescence staining used the following primary antibodies: anti-CTSL/major excreted protein and anti-CTSD from ABclonal Technology; anti-light chain 3 (LC3) B from Beyotime (Shanghai, China); anti-Sequestosome 1/p62 (SQSTM1/p62), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-lysosome-associated membrane protein 2 (LAMP2), anti-β-actin, anti-α-tubulin, and anti-Beclin 1, from Protein tech; and Peroxidase-Conjugated AffiniPure Goat Anti-mouse IgG and goat anti-rabbit IgG from Cwbio. Catalog and dilution information of these reagents are listed in Table 2.

Ginsenoside Rb1 (molecular weight = 1109, purity > 98%) was purchased from Shanghai Winherb Medical S&T Development (Shanghai, China). Oil Red O and commercial kits for detecting total cholesterol (TC), triglycerides (TG), cholesterol (CHOL), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were obtained from Biosino Biotechnology & Science Inc. (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for measuring mouse IL-6 and TNF-α were obtained from Beijing Expandbiotech Co., Ltd. (Beijing, China). Mouse MCP-1 and IL-1β ELISA kits and a kit for terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) were purchased from Roche Diagnostics GmbH (Mannheim, Germany). DAPI dihydrochloride staining reagent was provided by Beyotime Institute of Biotechnology (Beijing, China). A BCA protein assay kit, protease inhibitor cocktail and enhanced chemiluminescence kit were obtained from CWbiotech (Beijing, China). Mammalian protein extraction kits were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Primary antibodies were purchased from Abcam (Cambridge, UK), and secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. Dimethyl sulfoxide (DMSO) was from Sigma Aldrich (St. Louis, MO, USA).

RIPA buffer (Beyotime, Shanghai, China) was utilized for extracting total proteins from BMSCs and murine cartilage tissues. Quantification of proteins were achieved using a Bicinchoninic acid Protein Assay Kit (Beyotime). Protein samples (20 μg) were resolved with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen) [29 (link)]. After blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against Map3k2 (ab33918, 1:10000, Abcam), Aggrecan (13880-1-AP, 1:1000, Proteintech, Chicago, USA), collagen II (Col2, ab34712, 1:1000, Abcam), collagen X (Col10, ab182563, 1:1000, Abcam), matrix metalloproteinase 13 (MMP13, ab39012, 1:3000, Abcam), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS, ab41037, 1:250) and β-actin (ab6276, 1:5000, Abcam) overnight at 4°C. After that, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam) at room temperature for 2 hours. Eventually, protein signals were visualized using an enhanced chemiluminescence kit (Cwbiotech, Beijing, China) with an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).

To investigate the rTsTryp degrading or down-regulating expression of gut epithelium TJs proteins and related pathways, soluble proteins from Caco-2 monolayers pretreated with 20 μg/ml rTsTryp at 37°C for 2 h were analyzed by western blotting as described before [14 (link),37 (link)]. Briefly, Caco-2 cell monolayers were incubated with rTsTryp at 37°C for 2 h, cell proteins were collected, and the cell lysates were separated by 10% SDS-PAGE and transferred subsequently onto PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk in TBST for 1 h at 37°C, and cut into strips. Subsequently, the strips were probed overnight at 4°C with antibodies against ZO-1 (1:1 000, Servicebio, Wuhan, China), E-cad (1:200, Santa Cruz, USA), occludin (2 μg/ml, Santa Cruz), claudin-1 (2 μg/ml, Santa Cruz), PAR2 (1:1 000, Abcam, UK), p-ERK1/2 (1:1 000, Abmart, Shanghai), ERK1/2 (1:1 000, Abmart, Shanghai), GAPDH (1:1 000), and β-actin (1:2 000) (Servicebio). After washing with TBST, the strips were incubated with HRP -conjugated anti-mouse IgG or anti-rabbit IgG as secondary antibodies (1:10 000). Finally, the color of the strips was developed using an enhanced chemiluminescence kit (CWBIO, Beijing, China). The results for the above proteins were normalized using GAPDH as a loading control [4 (link),18 (link)].