Fig 1: Inflammatory priming is required for SFK activation to produce downstream signaling in the absence of receptor clustering.(A) CskAS BMDMs were incubated 12–16 hr in non-priming medium or in medium containing 25 U/ml IFN-?. Signal transduction after 3-IB-PP1 or zymosandep treatment was assessed by immunoblotting with antibodies specific to activating phosphorylation sites of DAP12, Syk, LAT, PLC?2, PLC?1, and Erk (arrow). Total Erk1 is shown as a loading control. (B) Signal transduction in the PI3K/Akt and JNK pathways was assessed by immunoblotting with antibodies specific for phosphorylated c-Cbl, PI3K, Akt (arrow), and JNK (arrow). Total JNK is shown as a loading control. NFAT1 activation was assessed by the faster migration of cellular NFAT1 upon dephosphorylation. See Figure 5—figure supplement 1 for further characterization of signaling in primed and unprimed cells.DOI: http://dx.doi.org/10.7554/eLife.09183.011