C myc antibody

Equal amounts of protein were resolved on an SDS-PAGE gel and subsequently transferred onto a polyvinylidene difluoride (PVDF; Millipore) membrane. The primary antibodies used were as follows: 1:2,000 α-Tubulin (ab4074); 1:1,000 14-3-3ε (CST #9635); 1:1,000 14-3-3 sampler kit (CST #9769T); 1:1,000 RIG-I (CST #3743); 1:2,000 c-Myc antibody (ThermoFisher MA1-980); 1:2,000 myc tag antibody (Bethyl A190-105A); 1:2,000 FLAG M2 (Millipore F1804); 1:1,000 VDAC1 (ab15895); 1:1,000 TBK1/NAK (CST #3504); 1:1,000 phospho-TBK1/NAK (CST #5483); 1:1,000 hnRNP M (sc-134360); 1:3,000 VP1 (Dako); 1:1,000 β-Actin (ab8224); 1:1,000 PARP (CST #9542); and 1:2,000 GFP (Roche 11814460001).

For in vitro ubiquitination assays, the HA-Ubiquitin plasmid was transferred into either PTBP1 knockdown or overexpressed AGS and HGC-27 cells. After 24 h, cells were incubated with 100 nM MG132 (Selleck, cat. no.: S2619) for 12 h. Then, the co-immunoprecipitation (Co-IP) assay was performed using the Pierce^TM Classic Magnetic IP/Co-IP Kit (Thermo Scientific, cat. no.: 88804). Briefly, cell lysates were combined with the c-Myc antibody (Thermo Scientific, cat. no.: PA5-85185) and treated overnight at 4 °C. On the next day, antigen/antibody complexes were bound to Protein A/G magnetic beads for 1 h at room temperature. Magnetic beads were washed twice with the immunoprecipitation lysis/wash buffer and once with water. After elution of antigen/antibody complexes, the WB with anti-Ubiquitin (Invitrogen, cat. no.: 701339) was performed. PTBP1 and c-Myc protein levels were also determined in whole-cell lysates (WCL).