Figure 4

Correlation between CD98-integrin association and CD98 heavy chain-light chain dimerization. A. NIH 3T3 cells (10⁷ cells/experiment) expressing wild type human CD98 heavy chain, CD98 heavy chain mutants (C109S or C330S) or control NIH 3T3 cells were lysed in 1% Brij 58 and then proteins were immunoprecipitated using 4F2 mAb. Cell lysates (left 4 lanes) and immunoprecipitates (right 4 lanes) were resolved in 8% SDS-PAGE, transferred to nitrocellulose membrane, probed with biotinylated KMI6 mAb (anti-mouse β1 integrin) followed by incubation with ExtrAvidin-HRP (Sigma) and developed using Renaissance Chemiluminescence Reagent (NEN Life Science Products, Boston, MA). B. HT1080 cells (10⁷ cells/experiment) were lysed in sodium carbonate buffer, pH 11 and fractionated in isopycnic discontinuous sucrose gradient as described in Materials and Methods. Fractions were analyzed by SDS-PAGE electrophoresis followed by Western blot analysis using biotinylated TS2/16 mAb or polyclonal anti-caveolin antibody, or adjusted to pH 7.5 and immunoprecipitated with 4F2 mAb and then blotted with biotinylated TS2/16 mAb. C. NIH 3T3 cells (10⁷ cells/experiment) expressing wild type human CD98 heavy chain, or mutant CD98 heavy chains (C109S or C330S) were cell surface biotinylated, lysed in sodium carbonate buffer, pH 11 and fractionated in sucrose gradients as described in Materials and Methods. Fractions were adjusted to pH 7.5 and immunoprecipitated using 4F2 mAb. Immunoprecipitated proteins were separated in 8% SDS-PAGE under non-reducing conditions and blotted onto nitrocellulose membrane. Blots were developed using ExtrAvidin-HRP (Sigma) and Renaissance Chemiluminescence Reagent (NEN Life Science Products, Boston, MA). For NIH3T3 cells, flow cytometry was used to demonstrate comparable levels of wild type CD98 (MFI = 27), C109S (MFI = 30), and C330S (MFI = 53) expression. Cells were 100% positive, with homogeneous peaks, well above background staining (MFI = 1). MFI=Mean Fluorescence Intensity. D. Relative distribution of CD98 wild type and C109S and C330S mutants in light (fractions 3–6) and dense (fractions 9–12) fractions from the Western blots shown in Fig. 4C. Densitometry was carried out using GeneGenius Bio Imaging System and analysis software, (Syngene, Frederick, MD). A typical experiment out of three performed is shown.