PLCγ/ERK/c-Fos, JNK/c-Jun, STAT, and SHP2 signaling from the MCSFR or GCSFR in Ba/F3 cells. (A) Ba/F3(GR,MR) cells withdrawn from IL-3 for 1 hour were cultured for an additional 30 minutes in the absence of cytokine (C), with M-CSF (M), or with G-CSF (G), together with no inhibitor or with a JNK (10 μM SP600125), MEK1/2 (10 μM U0126), or PLCγ (2 μM U73122) inhibitor, followed by Western blotting. (B) Ba/F3 cells were evaluated similarly after culture with no inhibitor or with a MEK1 (50 μM PD98059) or a general PLC (10 μM edelfosine) inhibitor. A vertical line has been inserted to indicate repositioned gel lanes. (C) Ba/F3(GR,MR) cells withdrawn from IL-3 for 1 hour were cultured for an additional 30 minutes in the absence of cytokine (C), with M-CSF (M), or with G-CSF (G), together with no inhibitor or with a JNK (10 μM SP600125), MEK1/2 (10 μM U0126), or PLCγ (2 μM U73122) inhibitor, followed by Western blotting (top panel). Ba/F3(GR,MR) cells were analyzed for P-STAT3 or STAT3 expression in IL-3, after removal of IL-3 for 1 hour, or after an additional 30 minutes in the absence of cytokine, with M-CSF, or with G-CSF (bottom panel). (D) Ba/F3 cells withdrawn from IL-3 were cultured for 30 minutes with no cytokine, M-CSF, or G-CSF. Total cellular protein extracts were then subjected to Western blotting for P-SHP2 Y542 and Y580, total SHP2, and tubulin.