(a) Fluorescence micrographs of 293T cells that were cotransfected with plasmids encoding Flag-TRAF6 and HA-BPLF1 (upper panel), NEMO and Flag-BPLF1 (middle panel), or IκBα and Flag-BPLF1 (lower panel). Sixteen hours post-transfection, signaling intermediates (column 2) and BPLF1 (column 3) were labeled using anti-Flag (red; for TRAF6 in upper panel; for BPLF1 in middle and lower panels), anti-BPLF1 (green), anti-NEMO (green), and anti-IκBα (green) Abs as indicated. Nuclei were visualized using TO-PRO-3 (blue, column 1). Column 4 and 5 show merged pictures from red and green channels without and with nuclear stain (blue), respectively. Scale bar 50 µm. (b) 293T cells were cotransfected with plasmids encoding either Flag-TRAF6 (upper panel) or His-NEMO (lower panel) together with HA-ubiquitin and BPLF1, BPLF1C61A, or A20. Expression of Flag-tagged proteins was assessed by immunoblotting (IB) with an anti-Flag Ab. TRAF6 and NEMO were precipitated (IP) from post-nuclear lysates and detected using an anti-TRAF6 or anti-His Ab, respectively. HA-tagged ubiquitin adducts were visualized on immunoblot using anti-HA Abs. (c) 293T cells were cotransfected with plasmids encoding HA-ubiquitin together with Flag-TRAF6 (left panel), His-NEMO (middle panel), or IκBα (right panel). IκBα-transfected cells were additionally treated with TNFα and the proteasome inhibitor MG132 for 2 hours before lysis. Twenty-four hours post-transfection, TRAF6, NEMO, and IκBα were precipitated from post-nuclear cell lysates and incubated in vitro with separately purified Flag-BPLF1, Flag-BPLF1C61A, or Flag-A20 proteins for 4 hours at 37°C. Flag-tagged DUBs were detected by immunoblotting with anti-Flag Ab and signaling intermediates were visualized using anti-Flag (TRAF6), anti-His (NEMO), or anti-IκBα Abs. HA-ubiquitin adducts were detected using an anti-HA Ab.