Ripa buffer

For the determination of changes in protein levels, treated cells were lysed in RIPA buffer supplemented with protease inhibitors (Promega), followed by protein quantification with Bradford reagent (Bio-Rad, Hercules, CA, USA). Total proteins from each sample were separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Bio-Rad) with a Transblot system (Bio-Rad). Proteins were detected with rabbit primary antibodies directed to survivin (Abcam, Cambridge, UK), PARP1 (Cell Signaling Technology, Danvers, MA, USA), N-cadherin (Thermo Fisher Scientific), cyclin B1 (Abcam), cyclin D1 (BD Pharmigen, Franklin Lakes, NJ, USA) or β-actin (Abcam) and secondary peroxidase-labeled anti-rabbit IgG. Blots were revealed with the EZ-ECL chemoluminiscent substrate (Beit-Haemek, Israel) and detected in a C-Digit Blot Scanner (LI-COR Biosciences, Lincoln, NE, USA). The quantification of protein bands was performed with the imageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA).

Lung and liver samples were homogenized with RIPA buffer (Promega, Madison, WI, USA) using a FastPrep‐24 5G Instrument (MP Biomedicals, CA, USA). Then, the supernatants were collected. Protein concentrations were measured by a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Then, equal amounts of protein samples were separated by 10% SDS-PAGE and then electro-transferred to polyvinylidene fluoride (PVDF) membrane. After blocking for 90 min with 5% nonfat dry milk at room temperature, the membranes were incubated overnight at 4 °C with specific primary antibodies. The next day, after incubating with secondary antibodies, the proteins were treated with ECL reagent (Pierce, WI, USA) for blot detection.

AGS and MKN45 cell lysates were collected using RIPA buffer plus RNase inhibitor (Promega), followed by incubation with the biotin-labeled RNA probe targeting the junction site of circ-MAT2B designed and synthesized by RiboBio at 37 °C for 2 h. Then, the washed streptavidin magnetic beads (Invitrogen) were added into above lysates and incubated for 30 min at 37 °C. After six washes, the immunocomplexes bound by circ-MAT2B probe were eluted for RNA extraction and qRT-PCR analysis.

Total cell lysates from PBMCs were prepared using a radio immune precipitation assay (RIPA) buffer (Promega, Madison, WI) with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined by DC protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of cell lysates (20 μg) were separated on 8% to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) precast gels and transferred to a polyvinylidene fluoride membrane (Millipore, Eschborn, Germany). Membranes were washed with Tris-buffered saline-Tween 20 (TBST), blocked in TBST containing 5% nonfat milk at room temperature for 1 hour, and then incubated overnight at 4 °C with following primary antibodies: mouse anti-β-act in antibodies (1:5000), mouse anti-TLR9 antibody (1:500), mouse anti-MyD88 (1:2000), rabbit anti-IRF7 antibody (1:1000), mouse anti-ISG56 antibody (1:500), and rabbit anti-MxA antibody (1:500). All these antibodies were purchased from Abcam, Inc. (Cambridge, MA). The membranes were then incubated for 1 hour at room temperature with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:5000, Abcam Inc., Cambridge, MA). Blots were developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA). Densitometric analysis was performed using ImageJ 1.44 software (National Institutes of Health, USA).