International Journal of Proteomics / 2013 / Article / Fig 2

Research Article

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

Figure 2

Degradation of WT-TCRα and KR-TCRα is dependent upon the E3 ubiquitin ligase Hrd1. (a) The various TCRα constructs were cotransfected with WT-Hrd1 or DN-Hrd1. Transfected cells were incubated with MG132 before lysis. Cellular lysates were immunoprecipitated with an anti-HA antibody and analyzed by Western blot using an anti-HA antibody. (b) 293T were cotransfected with a SMARTPool of siRNA against Hrd1 or negative control siRNA and vector, WT-TCRα, or KR-TCRα (HA-tagged). 24 hrs posttransfection, cells were treated with MG132 and lysed and levels of TCRα were determined by Western blotting with an anti-HA antibody. Levels of Hrd1 were determined by qRT-PCR.

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