LPS or PMA stimulates release of Mer ectodomain. Surface expression of Mer on J774 cells treated with 50 ng/mL LPS for 4 hours (A) or treated with 50 nM PMA for 45 minutes (B) was evaluated by flow cytometry. Expression on untreated cells at each time point is shown for comparison. (C) J774 cells were incubated in serum-free medium with or without 50 ng/mL LPS or (D) treated with 50 nM PMA for the indicated times. Mer and actin present in cell lysates and sMer released into the medium at each time point were analyzed by immunoblotting. (E) The carboxyl-terminal portion of cleaved Mer remains cell-associated. Elicited peritoneal macrophages from Mer^KD or wild-type mice were cultured overnight, then placed in serum-free medium and treated with 100 ng/mL LPS for the indicated times. Cells were lysed, and proteins from equal numbers of cells were separated by SDS-PAGE and immunoblotted with an antibody against the C-terminus of Mer. Membrane-bound Mer receptor (200 kDa), which is absent from Mer^KD macrophages, decreases with time of LPS treatment. The C-terminal 60-kDa fragment of Mer that accumulates with LPS treatment is indicated by an asterisk.