A, 293T cells stably expressing YFP-tagged TCRα were treated with MG132 (20μM) for the indicated time periods. TCRα-YFP-containing complexes were immunoprecipitated from NP40 lysis buffer-generated cell extracts and analyzed by immunoblotting. Where indicated, a fraction of the whole cell extract (WCE) was analyzed directly by immunoblotting. Asterisk indicates a deglycosylated TCRα species. LE, long exposure; SE, short exposure. B, As in A, except that siRNA transfected cells were treated with MG132 for 6h. C, The Bag6-TCRα complex is stable in the RIPA buffer. 293T cells expressing YFP-tagged TCRα were treated with MG132 (20μM) for 4h. Cells were lysed in either a NP40-containing lysis buffer or the RIPA buffer. Cell extracts were subject to immunoprecipitation and immunoblotting by the indicated antibodies. See also Figure S3