The proteasome activator PA28γ is a cellular partner for HTLV-1 p30. (A) 293T cells transfected with HA-p30 were immunoprecipitated with α-HA (3F10), and samples were analyzed by SDS-PAGE and silver staining. Specific bands were analyzed by mass spectrometry. (B) 293T cells were co-transfected with HA-p30 and Flag-PA28γ, and cell lysates were immunoprecipitated with the indicated antibodies and blotted with either HA or PA28γ antibodies to detect both FLAG-tagged and endogenous PA28γ. (C) HTLV-1 p30 does not interact with PA28α. 293T cells were transfected, immunoprecipitated, and immunoblotted with the indicated antibodies. (D) p30 expression does not alter PA28γ immunolocalization. Cells were transfected with GFP-p30 with or without FLAG-PA28γ. Immunofluorescence was performed after 48 hours. (E) p30 interacts within the first 50 amino acid residues of PA28γ. 293T cells were co-transfected with the indicated PA28γ mutant constructs along with p30 HA, and were immunoprecipitated and immunoblotted. (F) Alignment of the PA28γ and PA28α amino acid sequence. Swapping a 6 amino acid α helix region from PA28γ with those of PA28α abrogates p30 interaction with PA28γ in cotransfection experiments. (G) Nuclear localization of PA28γ(α21-26) is not altered. HeLa cells were transfected with PA28α, PA28γ, PA28γ(α21-26), and PA28γ(α1-34), fixed and stained with α-FLAG, and visualized by immunofluorescence.