Figure 3. PAD expression in APCs alone... | Rockefeller University Press

$Figure 3. PAD expression in APCs alone is not sufficient for citrullination of HEL; PAD activity can be detected in autophagosomes. (A and B) PAD2 (A) and PAD4 (B) mRNA was measured by RT-PCR and quantified using DNA standards. Data are presented as the mean of quadruplicate reaction replicates and are representative of at least two independent experiments. (C) PAD activity was measured biochemically in lysates of C3.F6.P4 cells after the addition of tetracycline. Data are the mean of triplicate reactions and are representative of three independent experiments. (D) Presentation of HEL or HEL peptide to Granny by C3.F6.P4 with or without tetracycline to induce PAD4 expression in normal culture conditions or after culture in DME without FCS (SS in the panel). Data are representative of three independent experiments. (E) PAD activity as measured by conversion of an artificial substrate was assessed in fractions of elicited PECs. The fractions are represented with numbers as follows: 1, whole cell lysate; 2, postnuclear supernatant; 3, nuclei; 4, second pellet enriched in mitochondria and peroxisomes; 5, light membrane fraction; 6, complex heavy fraction enriched in the ER; and 7, final autophagosome pellet. The negative control (Neg) contained substrate without protein. The data presented are pooled from three independent experiments. 20–60 mice were used in each experiment. (F) The fractions were analyzed by Western blot for LC3II enrichment. The data are representative three independent experiments. Error bars indicate SEM.$

Figure 3.

PAD expression in APCs alone is not sufficient for citrullination of HEL; PAD activity can be detected in autophagosomes. (A and B) PAD2 (A) and PAD4 (B) mRNA was measured by RT-PCR and quantified using DNA standards. Data are presented as the mean of quadruplicate reaction replicates and are representative of at least two independent experiments. (C) PAD activity was measured biochemically in lysates of C3.F6.P4 cells after the addition of tetracycline. Data are the mean of triplicate reactions and are representative of three independent experiments. (D) Presentation of HEL or HEL peptide to Granny by C3.F6.P4 with or without tetracycline to induce PAD4 expression in normal culture conditions or after culture in DME without FCS (SS in the panel). Data are representative of three independent experiments. (E) PAD activity as measured by conversion of an artificial substrate was assessed in fractions of elicited PECs. The fractions are represented with numbers as follows: 1, whole cell lysate; 2, postnuclear supernatant; 3, nuclei; 4, second pellet enriched in mitochondria and peroxisomes; 5, light membrane fraction; 6, complex heavy fraction enriched in the ER; and 7, final autophagosome pellet. The negative control (Neg) contained substrate without protein. The data presented are pooled from three independent experiments. 20–60 mice were used in each experiment. (F) The fractions were analyzed by Western blot for LC3II enrichment. The data are representative three independent experiments. Error bars indicate SEM.

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