The released intracellular contents of late apoptotic cells do not activate ERK1/2
A, the procedure used to separate late apoptotic and necrotic BUMPT cell suspensions into five distinct fractions is diagramed. Fractions 1–3 were derived from late apoptotic and necrotic cell suspensions corresponding to a cell to m ratio of 1:1, whereas Fractions 4 and 5 were derived from suspensions corresponding to a cell to m ratio of 4:1. B, serum-starved BM macrophages were exposed to late apoptotic cells (Fraction 1) and the 4 fractions (Fractions 2–5) from late apoptotic cell for 15 min alone or were stimulated with M-CSF for 15 min following a 30-min pre-exposure to these fractions. C, serum-starved BM macrophages were exposed similarly to necrotic cells (Fraction 1) or their fractions (Fractions 2–5) for 15 min alone. Nonadherent cells were removed by rinsing, and macrophage lysates were probed with anti-active ERK1/2 antibody. Equal loading was confirmed by Ponceau S staining of blotted proteins as well as probing for total ERK1/2 (supplemental Fig. S6). Late apoptotic and necrotic cells alone contributed no active ERK1/2, p38, JNK, or p90Rsk in these experiments.