In vitro citrullinated fibronectin has delayed migration by SDS-PAGE, altered antigenicity, but coats plastic normally. (A) Fibronectin incubated in buffer (FN), fibronectin incubated in buffer with peptidyl arginine deiminase (FNC), and fibronectin incubated in buffer with heat-inactivated peptidyl arginine deiminase (FNHI) were subjected to SDS-PAGE under reducing conditions followed by Coomassie Blue staining. Gel is representative of three experiments. Thick band at the bottom left of the gel shows the four indicated size markers combined in this 6% acrylamide gel. L, protein standard ladder. (B) FN and FNC were subjected to SDS-PAGE under nonreducing conditions followed by western blotting with the anti-fibronectin antibodies 5C3 or 4D1 (4D1 requires Arg107 for binding.). Blots are representative of three experiments. (C) Plastic wells were incubated with PBS, FN, or FNC followed by washing with PBS. Adhered protein was quantified using BCA reagent. Data shown are the average and standard error of 10 replicates from two independent experiments.
Shelef et al. Arthritis Research & Therapy 2012 14:R240 doi:10.1186/ar4083