Plasmin binding to RAW264.7 macrophages is reduced following incubation with EGTA.
(Left panel) Adherent cells (105/well) were washed with DPBS (3 ×). The media were replaced with cold MSFM alone or MSFM containing 0.2 to 8 μg/mL Lys-plasmin. Following incubation for 1 hour at 4°C, cells were washed (3 ×) with DPBS and membrane-bound plasmin activity determined as described in “Materials and methods.” Data represent the mean ± SE of 3 separate samples. (Right panel) Adherent cells (105/well) were washed with versine buffer (PBS/0.5 mM EDTA) at 4°C and then incubated with versine buffer containing 25 mM EGTA for 20 minutes. The EGTA was removed and cells were washed with versine buffer. The cells were incubated with versine buffer containing 25 mM EGTA, 0.1% bovine serum albumin, and Lys-plasmin (1.0 μg/mL) for 1 hour at 4°C. Membrane-bound plasmin (Pls) activity was determined as described in “Materials and methods.” The data represent the mean ± SEM of 3 replicate samples. The inset is a Western blot for annexin II of membranes isolated from Ctrl (control) and EGTA-treated cells.
