Fig 2: Differential gene expression of PAD2, PAD4, Ro60, and La induced by molecular stimulants ATP, LPS, TNF, IL-6, and IFNγ by quantitative PCR (qPCR). Graphs show the validation done by the comparative method (ΔCt). The expression level of each gene was normalized using 18S gen as invariant endogenous control. Asterisk on the top of the bars means that induced gene expression was significantly different with a p value of <0.0001 Student t-test.

Fig 3: PAD induction by stimulants. PAD2 detection by immunohistochemistry (brown stain). The expression of the enzyme is induced by the stimuli with different molecular triggers.

Fig 4: PAD activity. (a) In vivo PAD activity measured based on the citrullination of proteins in cell extracts using a direct ELISA assay with tissue extracts fixed onto polystyrene plates and measuring CCP/HRP antibody, this assay shows a significant increase by TNF (p < 0.0001). In vitro PAD2 activity under basal conditions in response to stimulation with different molecular stimulants; PADs were captured using anti-PAD2 or anti-PAD4 enzymes, then citrullination was detected using an exogenous reporter protein (fibrinogen) and measured with an anti-citrulline-HRP antibody by ACELIA and difference between stimulants and controls (b, c). (b) Significant differences of in vitro citrullination PAD2 dependent by the effect of all stimulants except IFNγ (p < 0.0001); similar results were obtained by PAD4, and bars marked with an asterisk compare control with a specific molecular stimulant by Student t-test (c).