Table 1. A list of UBE2F or UBE2M binding proteins-DC-D-23-00339R2
Description
H1299 cells were transfected with pIRES2-UBE2F (WT or C116A mutant) or pIRES2-UBE2M (WT or C111S mutant), along with empty pIRES2 vector as a negative control, using Lipofectamine 2000, and then selected with G418 for stable expression. Stable cells expressing above constructs were harvested individually from 20 x 150 mm plates each, then lysed in NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing inhibitors of protease and phosphatase. The cell lysates were incubated with Strepavidin Sepharose for 1 h at 4 °C. After washing, the bound proteins were eluted in 1 mg/ml Biotin for 30 min at 4 °C, and then incubated with S beads for 1 h at 4 °C. Finally, the UBE2F/2M binding proteins, were eluted by boiling in SDS sample buffer for 10 min, and then resolved in the PAGE gels. The sample-containing gels were cut after a short run with all the samples entering the running gels, and submitted to Harvard Medical School Taplin Mass Spectrometry core facility for protein identification. The results were provided in the Excel sheets by the Harvard MS core facility for all identified proteins in five samples.