10.1371/journal.pone.0022671.g006 Marco Thomas Marco Thomas Peter Lischka Peter Lischka Regina Müller Regina Müller Thomas Stamminger Thomas Stamminger Confirmation of UAP56 interaction domains in mammalian cells. Public Library of Science 2013 uap56 domains mammalian 2013-02-20 16:31:43 Figure https://plos.figshare.com/articles/figure/_Confirmation_of_UAP56_interaction_domains_in_mammalian_cells_/424548 <p>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<sub>hc</sub> =  immunoglobulin heavy chain; Ig<sub>lc</sub> =  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).</p>