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. 1999 Aug 31;96(18):10326–10331. doi: 10.1073/pnas.96.18.10326

Figure 5.

Figure 5

HLA-A2-GFP complexes can be detected in MIICs after subcellular fractionation by DGE in the presence of the lectin wheat germ agglutinin. (A) Postnuclear supernatant of Mel JuSo HLA-A2-GFP stimulated for 48 h with 200 units/ml of IFN-γ and cultured for 30 min with HRP was fractionated by DGE. Total protein concentration (squares) and enzymatic activity of HRP (diamonds) and β-hexosaminidase (triangles) were determined in alternate fractions. The peak positions of the lysosomes (L), late endosomes (E), the ER, and plasma membrane (PM) are indicated; early endosomes migrate around fraction 6. Every five fractions were pooled such that the lysosomal fractions (peak β-hexosaminidase activity) were in one pool. (B) The pooled fractions were lysed and split in two: one half was incubated on ice for 2 h (lanes A) and the other half was incubated at 37°C for 2 h (lanes B). MHC class I complexes were immunoprecipitated with W6/32 and visualized by Western blotting after separation by 10% SDS/PAGE using anti-heavy chain antibodies. Endogenous and HLA-A2-GFP complexes are present mainly at the plasma membrane (fractions 10 and 11), but a small amount of endogenous and HLA-A2-GFP complexes with stably bound peptide can be found in the lysosomes (fractions 3 and 4). (C) Sequential immunoprecipitation of the DGE fractions with anti-heavy chain antibodies. MHC class I heavy chains were immunoprecipitated with αHC and analyzed by Western blotting after separation by 10% SDS/PAGE using the anti-heavy chain mAbs HCA2 and HC10. HLA-A2-GFP and endogenous heavy chains can be retrieved from plasma membrane (fractions 10 and 11) and ER (fraction 7), but not from lysosomes and early and late endosomes (fractions 3–6). (D) Sequential immunoprecipitation of the DGE fractions with anti-MHC class II complex antibodies. MHC class II complexes were immunoprecipitated with Tü36 and analyzed by Western blotting after separation by 10% SDS/PAGE using anti-MHC class II α chain serum. MHC class II complexes are present at the plasma membrane (fractions 10 and 11), in the ER (fraction 7), and in MIIC (fraction 3 and 4) where class I complexes reside as well (B).