Figure 1
From: Stoichiometry and intracellular fate of TRIM-containing TCR complexes
The large αβTCR complex contains TRIM. (A) The single chain-tagged TCR (scTCR) was purified from digitonin-lysed 31.13scTCRβ cells using NP-sepharose affinity columns and free NIP for elution (lane 1). Pervanadate-stimulated Jurkat cells were lysed in digitonin and αβTCRs purified with anti-phosphotyrosine antibodies followed by phenylphosphate elution (lane 2). Membranes from Jurkat cells were lysed in digitonin (lane 3). TCRs were separated by BN-PAGE and immunoblotted against ζ. The marker protein was ferritin in its 24- and 48-meric forms (f1: 440 kDa, f2: 880 kDa). (B) 31.13scTCRβ (lanes 1 and 3) and Jurkat (lane 2) cell lysates were subjected to affinity purification on NP-sepharose columns. The purified proteins were separated on a reducing SDS-PAGE; TRIM and ζ were detected by immunoblotting. (C) NP-purified scTCRs were separated by 1D BN-PAGE and 2D non-reducing SDS-PAGE. Immunoblotting was done against scTCRβ, TRIM and ζ.