M-CSF preferentially activates PLCγ2, and phorbol ester induces ERK independent of SHP2. (A) Lineage-negative bone marrow (BM) cells were cultured with IL-3, IL-6, and SCF for 1 hour; removed from cytokine for 1 hour; and then cultured with no cytokine, M-CSF, or G-CSF for 5 minutes. Total cellular proteins were then subjected to Western blotting. (B) Ba/F3(MR,GR) cells removed from IL-3 for 1 hour were cultured with M-CSF or G-CSF for 0, 5, 15, or 30 minutes, followed by Western blotting. (C) Ba/F3(MR,GR) cells removed from IL-3 for 1 hour were loaded with Indo-1AM and then cultured with M-CSF or G-CSF, and the Indo-1 fluorescence at 395 nm (Ca²⁺ bound) and 500 nm (Ca²⁺ unbound) was assessed by FACS over a 7-minute period. An arrow indicates the time of cytokine addition. (D) Lineage-negative BM cells cultured with IL-3, IL-6, and SCF for 1 hour were removed from cytokine for 1 hour and then cultured with no cytokine in the absence or presence of 100 nM phorbol ester (TPA). U0126 (5 μM) or NSC-87877 (50 μM) was also included, where indicated.