Characterization of RNF122 expression in mammalian and E. coli cells. (A). HEK293T and HeLa cells were transiently transfected with RNF122-myc. At 24 h post-transfection, the cells were treated with 2.5 μg/mL MG132 or 50 μg/mL tunicamycin and harvested at the indicated time. The cell lysates were subjected to SDS-PAGE and analyzed by western blotting using anti-myc and anti-β-actin antibodies. (B). HEK293T cells were transiently transfected with RNF122-myc or with RNF122C92A-myc or RNF122C95A-myc mutants. At 24 h post-transfection, the cells were treated with 50 μg/mL MG132, harvested after 6 h, and then analyzed by western blotting. (C). GST-RNF122ΔTM or GST-RNF122-ΔTM mutant proteins were expressed in E. coli and purified by affinity chromatography. The proteins were verified by SDS-PAGE and western blotting using an anti-GST primary antibody. a: pre-induction; b: post-induction; c: effluent obtained after affinity chromatography; d: purified protein.
Peng et al. BMC Cell Biology 2010 11:41 doi:10.1186/1471-2121-11-41