Figure 3.

OTUD6B binds to and deubiquitinates the protein synthesis initiation complex. A, Sucrose fractionation of NSCLC cells extracts followed by immunoblotting. Lanes: 1, total cell lysate, 2, nuclear fraction, 3, postmitochondrial fraction, 4, postribosomal fraction, 5 ribosomes (see Supplementary Fig. S3 for Coomassie staining of representative gel). Ribosomal protein S6 has been used as a marker of ribosomes. B, Western blot analysis in immunoprecipitates obtained using an antibody against eIF4G and 50 μg of total cell lysates (indicated). A549 cells were transfected with either control siRNA or siRNA to OTUD6B. Please note that eIF4A appears as a single band or 2-3 bands whether it is run in minigels, or 20 cm long gels (respectively). OTUD6B also coimmunoprecipitated with eIF4G in H1299 cells (Supplementary Fig. S4). C, Western blot analysis of the indicated proteins in a representative 7mGTP pull-down assay or total cell lysate obtained from H1299 cells transfected with either control siRNA or siRNA to OTUD6B. Similar results were obtained in two additional experiments in H1299 and in A549 cells (one experiment). D, Left, Western blot analysis of H1299 cells transfected with a control plasmid (c) or with a plasmid encoding an OTUD6B fused at its C-terminal portion with an AH tag followed by 6 histidines at the indicated time points after transfection (24 and 48 hours). Right, Ni-NTA pull down of 200 μg of total cell lysates obtained from (24) and (c). E, immunoblots of 2D gels hybridized with an antibody against ubiquitin. Eluates from 7mGTP pull-down assays were run in 3 to 10 pH gradients (arrow), and subsequently run on a 10% SDS page gel, then transferred to nitrocellulose membranes.