FIGURE 3.

Peroxidasin, MPO, and EPO do not directly interact with collagen IV. a, PFHR-9 matrix was isolated after overlay with HEK293T cells transiently transfected with PXDN. The matrix (input) was collagenase or mock digested and the soluble and insoluble (pellet) fractions underwent SDS-PAGE under reducing conditions (with 50 mm DTT) and immunoblotted with anti-PXDN antibody. PXDN remained in the insoluble pellet despite collagenase digestion. b, in the same experiment as in a, the fractions were immunoblotted with anti-collagen IV α-2 NC1 domain antibody, which demonstrates that collagenase digestion solubilized the NC1 domain of collagen IV. After collagenase treatment, α-2 NC1 immunoreactivity is only present in the soluble fraction, whereas the mock digestion-insoluble pellet mimics input matrix with detection of partially solubilized full-length and degraded collagen IV. c, MPO and d, EPO also remain in the collagenase digest pellet as seen with PXDN in a.