International Journal of Cell Biology / 2014 / Article / Fig 3

Research Article

Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

Figure 3

MGP is crosslinked by DSP to vitronectin, fibronectin, and fibronectin III1-C polypeptide. (a) SDS-PAGE of DSP crosslinking reaction products of unlabeled proteins incubated with trace amounts of radioactive MGP. The presence of the crosslinker DSP, reducing agent 2-mercaptoethanol (2ME), is indicated by + or − in the appropriate lane. The protein present in the reaction is indicated above the lane; −, no added protein; VN, vitronectin; IIIC fibronectin III1-C polypeptide; FN, fibronectin; and OA, ovalbumin. (b) Gel filtration column separation of DSP crosslinked MGP and Fibronectin III1-C. Lines indicate 7 marker positions of 200, 116, 97, 66, 43, 31, and 14 kDa. DSP reacted sample containing labeled MGP and unlabeled III1-C (●); DSP reacted solution containing labeled MGP and unlabeled III1-C reduced with mercaptoethanol prior to column loading, (◯); labeled MGP alone (▵); Labeled MGP and unlabeled III1-C incubated with DSP vehicle (DMSO) (◊); DSP reacted solution containing labeled MGP alone (□). The exclusion volume was estimated by blue dextran elution, and total volume was estimated by phenol red elution. The column was run with 4 M guanidine containing buffer to disassociate noncovalent interactions (see methods).