Mini protein tgx precast protein gels

Three biological exosome samples (20 μg/sample) were separated on 4–15% mini-protein TGX precast protein gels (Bio-Rad) and subsequently stained with Coomassie Blue, then each sample lane was excised and digested with trypsin and spiked with 0.2 pmol of ADH peptides (YEAST Alcohol dehydrogenase 1) at the Mass Spectrometry Facility at the University of Massachusetts Medical School. The samples were then injected into Orbitrap Fusion Lumos Mass Spectrometer (Thermo Fisher Scientific) in technical triplicates for label-free quantitation (LFQ) analysis. The data were searched against Swiss-Prot Mouse protein database using Mascot search engine through Proteome Discoverer software. The data was exported and normalized as intensity-based absolute quantification (iBAQ) quantitative values in Scaffold (version Scaffold_4.10, Proteome software). The selected parameters for protein identification were the following: Protein Threshold >95%; minimum 3 peptides per candidate protein; Peptide Threshold >90%; >1 × 10⁵ iBAQ value in at least one of samples. The iBAQ value of the housekeeping protein ADH was used for normalization of biological replicates.

After the exposure, intestinal organoids were lysed in 100 μL RIPA buffer and the extracted proteins were denatured using SDS loading buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.04% bromophenol blue, and 100 mM β-mercaptoethanol) at 95 °C for 5 min. For each sample, 10 μg protein was loaded and separated on 10% or 12% mini-protein TGX precast protein gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, USA). The membranes were incubated overnight at 4 °C with appropriate primary antibodies, subsequently with specific secondary antibodies and visualized using the enhanced chemiluminescence reagent (Thermo Scientific). The following antibodies were used: Rabbit anti-alpha-TUBULIN, mouse anti-E-cadherin, and rabbit anti-NF-κB p65 were from Abcam; rabbit antibodies against p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, MLC, phosphor-MLC, phospho-NF-κB p65, STAT1, and phospho-STAT1 (Tyr701) were from Cell Signalling Technology; mouse anti-CLDN-2 and rabbit anti-ILDR-1 were from Invitrogen; anti-mouse/rabbit IgG horseradish peroxidase-linked secondary antibodies were from Cell Signalling Technology. Original western blot images were included in Supplementary Figures 7, 8, and 9. Densitometric quantification analyses of the western blots were performed using the Image J software.