Figure 3
Calcium-induced PAD4 activation dissociates PAD4-NCF1 complexes and inhibits ROS generation. (a) Citrullination of neutrophil proteins detected by anti-citrulline antibody F95 in cells stimulated with 1 µM ionomycin (in medium with 2 mM Ca2+), 100 nM PMA, or 10 µM DPI, as indicated. (b) Upper panel, anti-NCF1 immunoblot of anti-PAD4 immunoprecipitates from resting neutrophils (lane 1), or cells treated with the indicated stimuli. Lower panel, anti-PAD4 immunoblot of the same samples. (c) Confocal images of neutrophils treated with 1 µM ionomycin (in medium with 2 mM Ca2+) and then stimulated with 25 nM PMA and stained with anti-PAD4 (green) or anti-NCF1 (magenta). The last panel shows the co-location of pixels of the two colors. (d) Oxygen consumption as a measure of ROS generation by neutrophil stimulated by 100 nM PMA, first treated medium alone or with 1 µM ionomycin, as indicated. The data represent the results of three independent experiments.
