Confirmation of UAP56 interaction domains in mammalian cells.

Coimmunoprecipitation analyses to confirm the interaction of endogenous UAP56 and REF (A) and to map the UAP56-domains required for (B) REF- or (C) pUL69-interaction. (A) HEK293T cells were either mock transfected (lanes 1 and 2) or cotransfected with plasmids encoding FLAG-tagged UAP56 and myc-tagged REF (lane 3). The expression of endogenous proteins and proteins after transfection was controlled by Western blot analysis using an α-UAP56 antibody (anti-BAT1) (A, upper panel) and an α-REF antibody (A, middle panel). Immunoprecipitation was performed using an α-UAP56 antibody followed by detection of coprecipitated proteins with α-REF antibody (A, lower panel). The position of detected proteins is indicated on the right of each panel (UAP56, FLAG-UAP56, REF, myc-REF). Lysates of lane 1 were treated with RNase in order to exclude a bridging of proteins by RNA. (B) HEK293T cells were transfected with plasmids encoding either myc-tagged-UAP56 variants (lanes 1–9, as indicated) or myc-tagged REF (lanes 10 and 11). The correct expression of proteins after transfection was controlled by Western blot analysis using an α-myc antibody (B, upper panel). Expression of REF (either endogenous or transfected) was also verified by Western blot analysis using an α-REF antibody (B, middle panel). Immunoprecipitation was performed using an α-REF antibody followed by detection of cotransfected proteins with α-myc antibody (B, lower panel). The position of detected proteins is indicated on the right of each panel (Ig_hc =  immunoglobulin heavy chain; Ig_lc =  immunoglobulin light chain). (C) HEK293T cells were co-transfected with a plasmid encoding pUL69 in combination with vectors coding either for β-galactosidase alone or for fusions of UAP56 with β-galactosidase (as indicated). Expression of β-galactosidase fusion proteins and pUL69 was confirmed by Western blot experiments (C, upper and middle panel, respectively). Immunoprecipitation was performed using an anti-β-galactosidase antibody followed by the detection of coprecipitated proteins using an α-pUL69 antibody (C, lower panel).