Figure 2

Generation and characterization of A201 cell lines expressing CD4-WT and dimerization mutants. (A) A201 CD4-WT, A201 CD4-E91K, E92K and A201 CD4-Q344E cell populations were stained with the anti-CD4 mAb OKT4 and a FITC-conjugated secondary Ab. CD4 expression levels at the cell surface were determined by flow cytometry. The A201 cell line was used as a negative control. (B) A comparison of CD4 cell surface expression in A201 CD4-WT high cell population and PHA-activated PBMC is shown. (C) Flow cytometric analysis of CXCR4 cell surface expression in the different selected A201 CD4 cell populations and PHA-activated PBMC was performed after staining the cell surface with the anti-CXCR4 mAb 12G5 and a FITC-conjugated secondary Ab. (D and E) Cell lysates from the different A201 CD4 cell populations were normalized for total CD4 amounts and then immunoprecipitated with the anti-CD4 mAb OKT4. Immunocomplexes were then resuspended in non-denaturing Laemmli buffer and separated by SDS-PAGE. Immunoblot analysis was then carried out as described in Figure 1. The A201 cell line was used as a negative control. Panels of the same section show separated lanes of the same gel. The lower panels in E represent a shorter exposure of the same membrane, showing unsaturated bands of CD4 monomers. These results are representative of 3 independent experiments.