PADI4 Antibody (4D8)

Western Blot: PADI4 Antibody (4D8) [H00023569-M01] -

Western Blot: PADI4 Antibody (4D8) [H00023569-M01] - Release of peptidyl arginine deiminases (PADs) into the supernatant during NETosis, & detection of enzymatically active PADs. A, DNA release in peripheral blood neutrophils from healthy donors that were left unstimulated or stimulated with 25 mM phorbol myristate acetate (PMA) was assessed for up to 240 minutes after stimulation. Bars show the mean ± SD of 8 samples per group. B, For isolation of neutrophil extracellular traps, supernatants (SN) of unstimulated (unst.) or stimulated (stim.) neutrophils were collected. Subsequently, the cells were washed 3 times with RPMI medium (W1–W3), & stimulated cells were incubated in the absence or presence of DNase I; unstimulated cells treated with DNase I served as a control. Bars show the mean ± SD DNA concentration (conc) in 7 samples per group. C, Proteins (PAD2, PAD4, & neutrophil elastase [NE]) were precipitated from the same supernatants of unstimulated or stimulated cells as described in B & analyzed by Western blotting. Citrullinated proteins (citr.prot.) were detected using chemical modification & anti–modified citrulline antibody. PAD2 & PAD4 antibodies were tested for cross‐reactivity using human (hu) recombinant PAD4 (rPAD4) (250 ng) & human skeletal muscle tissue lysate (15 μg). One representative blot of 4 independent experiments is shown for each group. D, PAD activity in supernatants of unstimulated & stimulated cells was compared. Symbols represent individual donors (n = 10); bars show the median. ∗ = P < 0.05; ∗∗ = P < 0.01, by Wilcoxon's matched pairs signed rank test. AFU = arbitrary fluorescence units; NS = not significant. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26245941), licensed under a CC-BY license. Not internally tested by Novus Biologicals.