Figure 6.

Association and colocalization of NKAP with FADD. (a) 293T cells were transfected with HA or HA-NKAP expression plasmids, with either empty vector or FLAG-FADD. The next day, cell extracts were generated and subject to immunoprecipitation with anti-HA. Samples of extracts (WCE) and immunoprecipitates (IP) were analyzed by western blots with either anti-FLAG to examine FADD co-precipitation or anti-HA to assess immunoprecipitation. (b) NIH3T3 cells were transfected with either HA or HA-NKAP, in addition to FLAG-FADD expression plasmids. The next day, cells were fixed, permeabilized and stained with rabbit anti-FLAG and mouse anti-HA. DAPI was used to delineate nuclei. In the bottom panel, FLAG and HA are overlaid. Scale bar: 20 μm. (c) Western blot analysis of protein expression in FADD^−/− MEFs, while exogenous NKAP were expressed alone or co-expressed with FADD. Data are representative of three independent experiments. (d) Effect of co-expression with FADD on NKAP expression was studied by immunofluorescence microscopy. FADD^−/− MEFs were transiently transfected with HA-NKAP expression plasmids in the absence or presence of FLAG-FADD. At 48 h after transfection, cells were fixed and subjected to immunostaining for analyses. Scale bar: 10 μm