FIG. 6.
Interaction of various Nef alleles with NAK in Tg mice. IP, immunoprecipitation. (A) Total protein extracts from thymocytes (250 μg) and macrophages (100 μg) from CD4C/HIV-Nef Tg mice expressing the indicated Nef alleles and from their non-Tg littermates were incubated with rabbit anti-Nef polyclonal antibodies overnight and subjected to an IVKA using 10 μCi of [γ-32P]ATP for 5 min at room temperature. The phosphorylated proteins were next separated by SDS-PAGE and visualized by autoradiography. (B) Thymocytes from CD4C/HIV-NefJR-CSF, CD3C/HIV-Nef039, and CD4C/HIV-NefNL4-3(WT) Tg and non-Tg mice were metabolically labeled with [35S]methionine. Lysates were immunoprecipitated with rabbit polyclonal anti-Nef antibody produced in our laboratory. The 35S-labeled proteins were separated by SDS-PAGE and visualized by autoradiography. Note the ability of the polyclonal anti-Nef antibody used to immunoprecipitate the NefJR-CSF proteins effectively.