A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway
Figure 1
KR-TCRα is ubiquitinated and degraded by the proteasome. (a) HEK293T cells were transfected with a vector expressing WT-TCRα, or a vector expressing KR-TCRα. Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCRα was analyzed by Western blot analysis using an anti-ubiquitin antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). (b) HEK293T cells were cotransfected with 6XHis-Ub and either a vector expressing WT-TCRα or KR-TCRα. Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCRα was analyzed by Western blot analysis using an anti-His antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). Molecular weight markers (kD) of marker proteins are indicated.
