Fig. 3.

Inflammatory, but not apoptotic, granulocyte death leads to citrullination of fibrinogen. a Western blot of supernatants of trans retinoic acid (ATRA)/HL60 cells treated with indicated death-inducing stimuli and cultured in Hanks balanced salt solution (HBSS)/fibrinogen/5 mM calcium for 24 hours. Purified citrullinated fibrinogen and fibrinogen (without added ATRA/HL60 cells) were run as positive and negative controls, respectively. These lanes were not adjacent on the original gel. Blots were stripped and reprobed with anti-fibrinogen antibody as a load control. b Western blot of supernatants of ATRA/HL60 cells treated with indicated death-inducing stimuli and incubated in HBSS with or without fibrinogen and 2 mM calcium for 24 hours. c Western blot of primary human neutrophil lysate incubated with fibrinogen/calcium for indicated times. Peripheral blood neutrophils (PMN) alone (PMN alone) neutrophil lysate without fibrinogen, was not adjacent on the original gel. Western blots of time (T) = 0 ATRA/HL60 cell pellet or soluble fraction of ATRA/HL60 cells treated with four cycles of freeze thaw and then incubated for 24 hours and probed with anti-peptidylarginine deiminase (PAD)2 antibody (d) or anti-PAD4 antibody (e). f Western blot of soluble fraction of ATRA/HL60 cells treated with indicated death-inducing stimuli and cultured in HBSS/fibrinogen/5 m M calcium for 24 hours and probed with anti-PAD2 antibody (f) or anti-PAD4 antibody (g). PMA phorbol 12-myristate 13-acetate, IONO Ionomycin, GAPDH glyceraldehyde-3-phosphate dehydrogenase