Figure 6.

Association of lectin-deficient Crt with members of the peptide loading complex. (A) The indicated cell lines were lysed in digitonin lysis buffer and then subjected to immunoisolation with either anti-tapasin antiserum (lanes 1) or preimmune serum (lanes 2). Immune complexes adsorbed on protein A beads were dissociated with 100 μM of the C-terminal tapasin peptide used to raise the antiserum. Eluted proteins were separated by SDS-PAGE and immunoblotted with antibodies directed against Crt, ERp57, and the TAP peptide transporter. Note that the high background in the Crt panel is due to residual Ig heavy chain from the anti-tapasin antiserum (50 kDa), which migrates just below Crt (60 kDa). (B) The indicated cells were radiolabeled with [³⁵S]Met for 30 min, incubated on ice for 10 min with 20 mM N-ethylmaleimide in PBS, lysed in digitonin lysis buffer, and then subjected to immunoisolation with anti-tapasin antiserum. Isolated proteins were resolved on a nonreducing 10–15% gradient SDS-PAGE gel.