Cofilin exists as an oligomer in vivo and in vitro.
A) Human washed platelets (4×108/ml, 0.4 ml) and endothelial cells (0.8–1×106 cells/20 µl) were incubated with DMSO (1 µl) or membrane-permeable, non-cleavable, homobifunctional, maleimide cross-linkers (BMOE and BMH) at a final concentration of 1 mM and 0.2 mM, respectively. The cell lysates were immunoblotted with anti-cofilin antibody. A cofilin oligomer (∼65 kDa; indicated by asterisk) was observed in BMOE and BMH but not in DMSO-treated platelets and endothelial cells. B) Recombinant His-cofilin at concentrations ranging from 10 µM to 40 µM was incubated with BMOE (two fold molar excess of cofilin) or treated with DMSO for one hour at room temperature. BMOE- and DMSO-treated cofilins were subjected to SDS-PAGE and analyzed by coomassie blue staining. C) The cross-linked platelet and endothelial cells lysates (as described in A) were immunoblotted with anti-ADF antibody. A faint band (∼65 kDa) was observed in lysates of BMOE and BMH but not in DMSO-treated cells. D) Endothelial cells (0.8–1×106 cells/20 µl) were incubated with DMSO (1 µl) or BMOE (1 mM). For formaldehyde cross-linking, endothelial cells (1×106 cells/ml) were treated with formaldehyde at a final concentration of 1%. The cell lysates were subjected to SDS-PAGE on a gradient gel (4–15%) and were then immunoblotted with anti-cofilin antibody. A cofilin oligomer (∼65 kDa; indicated by asterisk) was observed in BMOE- and formaldehyde-treated cells, but not in DMSO-treated endothelial cells. Proteins were detected by fluorescence imaging of secondary antibodies labeled with infrared dyes.