Anti hsp70

Wild-type (WT) Salmonella enterica serovar Typhimurium LT2, described by Li et al. (2007) (link), was used in this study. The ΔsopB mutant strain was constructed using the λ-Red recombination system (Datsenko and Wanner, 2000 (link)). The Henle-407 human intestinal epithelial cells and RAW 264.7 macrophages were purchased from the American Type Culture Collection (ATCC). All cells were cultured in antibiotic-free Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C under a 5% CO₂ atmosphere.
Rabbit anti-phospho-Akt and rabbit anti-Akt antibodies were purchased from Cell Signaling Technology (Beverly, CA, United States). Rabbit anti-CD9, anti-CD63, anti-HSP70 and a secondary anti-rabbit antibody were purchased from System Biosciences (Bay Area, CA, United States). The ExoRNeasy Serum/Plasma Maxi Kit was purchased from Qiagen (Valencia, CA, United States).

The total proteins content in the iPSCs was extracted by a RIPA kit (Beyotime, Shanghai, China), and the protein samples of the sEVs were detected directly without extracted by a RIPA kit. The total amount of protein was detected by bicinchoninic acid method (BCA). All the samples were loaded in equal amount (15 μg), and then separated by a 10% SDS‐polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Beyotime, Shanghai, China). The membrane was blocked with 5% bovine serum albumin in TBST (Beyotime, Shanghai, China) and incubated overnight with primary antibodies. The blots were then washed with TBST, incubated with anti‐rabbit or anti‐mouse secondary antibodies (Cat. No. S0001 and S0002. Affinity Bioscience, OH, USA) and detected with the ECL‐2 reagent (Beyotime, Shanghai, China). The primary antibodies that were used in this study were anti‐HSP70, anti‐CD63, anti‐TSG101, anti‐calreticulin, and anti‐β‐actin which were all purchased from System Biosciences (SBI, Palo Alto, CA, USA) or Abcam (Cambridge, UK). Detailed information of antibodies was shown in the Table S1.

We isolated the serum exosomes as previously described (Lv et al., 2017 (link)). We first performed differential centrifugation (1000 g for 10 min at 4°C, and 16,500 g for 30 min at 4°C) to remove the cell debris, followed by ultrafiltration using 0.22 μm filters. We then performed ultracentrifugation (120,000 g for 2 h) to obtain exosome pellets.
Transmission electron microscopy (TEM) combined with immunogold labeling was employed to visualize exosomes (Melo et al., 2015 (link)). The exosome pellets were first suspended and dropped onto 200 mesh formvar carbon-coated nickel grids, followed by incubation with 50 mM glycine. After being blocked with 5% bovine serum albumin (BSA), the samples were incubated with rabbit anti-human antibodies (anti-CD9 (SBI, United States), anti-CD63 (SBI, United States), anti-Hsp70 (SBI, United States) and anti-Calreticulin (Abcam, United States)). The samples were then incubated with the goat anti-rabbit secondary antibody conjugated with protein A-gold particles (10 nm) (Bioss, China), followed by negatively stained with 3% phosphotungstic acid for 10 min. The exosome-containing grids were air-dried and observed using a JEM-1400 TEM (JEOL, Japan). In addition, we analyzed the exosome size distribution using nanoparticle tracking analysis (NTA, Malvern, United Kingdom) according to the manufacturer’s instructions.

Protein lysates from colon tissue and MDE were separated and transferred onto a polyvinylidene difluoride (PDVF) membrane. The membranes were probed with antibodies, and detected using enhanced chemiluminescence detection. Primary antibodies were as follows: anti CD81 (1:1000; Cosmo Bio, Tokyo, Japan), anti HSP70 (1:1000; SBI System Biosciences, Palo Alto, CA, USA), anti-CD9 (1:1000; SBI System Biosciences, Palo Alto, CA, USA), rabbit anti TGF-β1 (Abcam, Cambridge, MA, USA), and rabbit anti β-actin (Abcam, Cambridge, MA, USA). The secondary antibody was horseradish per-oxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (1:3000; Cell Signaling Technology, Danvers, MA, USA). Quantification was performed using NIH-Image software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2018).

Cells were lysed in SDS, separated by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were probed with antibodies and detected using enhanced chemiluminescence detection. Primary antibodies were as follows: anti-β-catenin (1:2000; Sigma Aldrich, St Louis, MO, USA), anti-β-actin (1:1500; R&D Systems, Minneapolis, MN, USA), anti-collagen Iα1 (Affinity Bioreagents, Golden, CO, USA), anti-DNMT1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-PTEN (1:1000; Epitomics, Burlingame, CA, USA), anti HSP70 (1:1000; SBI System Biosciences, Palo Alto, CA, USA), and anti CD81 (1:1000; Cosmo Bio, Tokyo, Japan). The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (1:3000; Cell Signaling Technology).
Quantification was performed using NIH-Image software (http://rsb.info.nih.gov/nih-image/download.html).