FIG. 1.

Role of the peptide-loading complex in m4/gp34-K^b association. (A) Effects of peptide-loading complex on the association of m4/gp34 with K^b molecules in the context of MCMV infection. TAP^−/−, tapasin^−/− (Tpn^−/−), or β₂m^−/− fibroblasts or normal MEFs from C57BL/6 mice were infected with wild-type MCMV (MOI = 20) and labeled overnight with ³⁵S. The cells were lysed in NP-40 lysis buffer and precleared with normal rabbit serum, followed by sequential IP with the polyclonal antiserum anti-p8 to precipitate K^b and its associated molecules and with antiserum 8139 to precipitate non-K^b-associated free m4/gp34. (B) m4/gp34-K^b complexes associate with tapasin. IFN-γ-treated B6 MEFs or K^bD^b−/− fibroblasts were infected with either wild-type or Δm4 MCMV for 3.5 h and then labeled with ³⁵S for 2 h. The cells were lysed in digitonin lysis buffer. Tapasin was immunoprecipitated from the precleared lysates. A control IP was performed with normal rabbit serum (NRS). The immune complexes were dissociated by boiling in 1% SDS for 10 min, resuspended in 1% NP-40 lysis buffer, and subjected to secondary IP against m4/gp34. Samples were treated with endo-H and analyzed by SDS-PAGE.