Active proteases are increased in conditioned medium (CM) from LRP-1–deficient cells. (A,B) CM from PEA-10, MEF-2, and B-41 cells was analyzed by ABPP. The probes used are specific for active serine hydrolases. (C) CM from PEA-10, MEF-2, and B-41 cells was analyzed by immunoblot using C1r-specific antibody (top panel). The corresponding cell extracts were immunoblotted using antibody specific for tubulin to confirm that the CM was collected from an equivalent number of cells (bottom panel). (D) ¹²⁵I-labeled active C1r was incubated for 1 hour with or without C1-INH. Active C1r is shown by the connected arrows. The complex of the C1r light chain with C1-INH (C1r-C1INH) migrates as the low mobility band. (E) ¹²⁵I-C1r or ¹²⁵I-C1r-C1-INH was incubated for 3 hours at 37°C with PEA-10 or MEF-2 cells. Receptor-associated protein (RAP; 0.5 μM) was added to some cells during the incubation. Cells were washed and treated with pronase to remove cell surface-associated ¹²⁵I-C1r. Radioactivity associated with the cell pellets was determined. Error bars represent SD.