Mini protean tgxtm stain free gel

To identify the role of nucleic acids or proteins in the transfer of EV-mediated TIG-R, TIG-R EVs (5 μg) were treated as previously described [24 (link),25 (link)] with minor modifications. Briefly, 2.5 μL of DNase I (1 U∙µL⁻¹, Zymo Research, Irvine, CA, USA), Sau3AI (4000 U∙mL⁻¹, New England Biolabs, Ipswich, MA, USA), or proteinase K (20 mg∙mL⁻¹, Thermo Fisher Scientific, Waltham, MA, USA) were treated with TIG-R EVs (5 μg) for 2 h at 37 °C according to the manufacturers’ instruction. The resulting EV fractions were characterized by 1% agarose gel electrophoresis, 12% Mini-PROTEAN® TGX^TM Stain-Free gel (Bio-Rad, Hercules, CA, USA) electrophoresis, and imaging analysis. The resulting samples were used to analyze the transference of TIG-R to the TIG-S AB strain.

Total EV proteins (10 µg) derived from TIG-S (ATCC 19606) and TIG-R AB were electrophoresed onto 12% Mini-PROTEAN® TGX^TM Stain-Free gel (Bio-Rad, Hercules, CA, USA) with Precision Plus Protein^TM Standards (Bio-Rad, Hercules, CA, USA). After staining gels with Brilliant Blue R (Sigma-Aldrich, Saint Louis, MO, USA) individual protein bands of new appearances or high intensity in TIG-R EVs compared with those from TIG-S were further subjected to in-gel MALDI-TOF/peptide mass fingerprinting (PMF) analysis using Microflex LRF-20 (Bruker Daltonics, Billerica, MA, USA). Spectra were acquired from 1000 shots per spectrum in the 700–4000 m/z range and calibrated by two-point internal calibration using trypsin auto-digestion peaks (m/z = 842.5094 and 2211.1040). The peak list was generated using flexAnalysis 3.0 (Bruker Daltonics, Billerica, MA, USA). Finally, the proteins were characterized using the MASCOT search engine (Matrix Science Inc., Boston, MA, USA) and UniProt database ([26 (link)]; https://www.uniprot.org; accessed on 13 February 2023).

Both a nanoparticle analyzer (NTA) and transmission electron microscopy (TEM), NanoSight NS300 (Malvern Panalytical, Malvern, UK) and Talos L120C TEM (Thermo Fisher Scientific, Waltham, MA, USA), respectively, were used to characterize the total particle concentration, size distribution, and morphology of EVs without disrupting their structure [20 (link),21 (link)]. The protein-based quantification of EVs was performed using Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) [22 (link)] with a SPECTROStar^® Nano (BMG LABTECH GmbH, Ortenberg, Germany). The protein components of EVs were characterized by 12% Mini-PROTEAN® TGX^TM Stain-Free gel (Bio-Rad, Hercules, CA, USA) electrophoresis and imaged through the ChemiDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and Image Lab Software (ver.5.2.1, Bio-Rad, Hercules, CA, USA) as described [23 (link)]. One representative of the three is shown.