FIGURE 1.
PML interacts with ASC. A, HEK293T cells were transfected with plasmids encoding empty vector green fluorescent protein (EV-GFP), PMLI-GFP, and/or ASC as indicated. 48 h post-transfection, cells were lysed, and 50 μl of whole cell lysate was kept for analysis. The remainder was immunoprecipitated (IP) with GFP-TRAPTM beads for 1–2 h at 4 °C. Whole cell lysates and immunoprecipitated samples were analyzed by Western blotting using anti-GFP and anti-ASC (AL177) antibodies. GFP pulldown resulted in the precipitation of PML-GFP (1st and 2nd lanes, 3rd panel) as expected and also ASC (1st lane, top panel). B, PML truncations were generated to identify the domains involved in the interaction with ASC. C, HEK293T cells were transfected with plasmids encoding ASC and EV-GFP or PML truncations as indicated. Proteins were precipitated as in A and analyzed by Western blotting. GFP pulldown resulted in the precipitation of ASC with PML(1–223), PML(1–491), and PML-FL. C, top panel, * denotes nonspecific band. Results presented are representative of four (A) and three (C) independent experiments. IB, immunoblot.