Figure 1.
PAD expression in human primary neutrophils and autoantigen recognition by RA sera in control and ionomycin-activated neutrophils. A. PAD2, 3 and 4 are expressed in human neutrophils. Samples from freshly isolated neutrophils were analyzed by immunoblotting with antibodies against human PAD2, PAD3 and PAD4. B, C. Primary human neutrophils in HBSS containing 2 mM CaCl2 were incubated in the absence (−) or presence (+) of 1 µM ionomycin for 4 hrs at 37°C. Samples were analyzed by electrophoresis on 13% SDS-polyacrylamide gels and immunoblotted using an AMC antibody (B) or anti-CCP positive sera from RA patients (C). Data from 4 representative sera are shown in C. The unfilled arrows denote antigens detected in non-stimulated neutrophils, filled arrows mark antigens generated upon neutrophil activation and the asterisk denotes the 47kDa species that was further analyzed by mass spectrometry.