PLP2 is required for the export of a K3•MHC-I complex from the ER.

<p>(<b>A</b>) Validation of the cell lines used for the experiment. FLAG-K3 and PLP2 were immunoprecipitated from the indicated [³⁵S]-methionine pulse-labelled cell types, resolved by SDS-PAGE and analysed by autoradiography. (<b>B</b>) K3-bound MHC-I remains EndoH-sensitive in the absence of PLP2. The indicated cell types were [³⁵S]-methionine pulse-labelled for 12 min, chased for the indicated times and solubilised in 1% digitonin. FLAG-K3 was immunoprecipitated using the anti-FLAG M2 mAb, dissociated in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10 and then either EndoH (+) or mock digested (−) before the samples were resolved by SDS-PAGE and analysed by autoradiography. (<b>C</b>) Loss of PLP2 does not affect the normal MHC-I synthetic pathway. Free [³⁵S]-methionine labelled MHC-I molecules were immunoprecipitated from the post-M2 supernatants (from B) using the W6/32 mAb, EndoH (+) or mock digested (−), and analysed by SDS-PAGE and autoradiography. (<b>D</b>) PLP2 co-immunoprecipitates with K3 and K5. FLAG-K3 and K5 were immunoprecipitated from digitonin lysates of HeLa cells using the anti-FLAG M2 mAb, and co-immunoprecipitated PLP2 detected by immunoblot.