Fig. 4

B16F0 melanoma cells transduce non-canonical signals in response to IL-12 stimulation by phosphorylating Akt on serine 472/473. Following a 12-hour preconditioning in the absence of FBS and IL12, B16F0 (top panels) and 2D6 (bottom panels) cells were stimulated with 7, 100, and 300 ng/mL IL-12 for 3 hours and assayed for phosphorylation of Akt (a) and STAT4 (b) by flow cytometry. Background levels of fluorescence associated with phosphorylated Akt and STAT4 in unstimulated cells are represented by the horizontal dotted lines (95% of population below), while the vertical lines indicate background fluorescence associated with IL12RB2 expression in no stain controls (95% of population with lower values). Results for three independent experiments with 3 biological replicates in each are summarized in panels (c) and (d), with p-values for pairwise comparisons indicated. Activation of Akt by IL-12 (100 ng/mL) in B16F0 was probed using antibodies that recognized phosphorylation at serine 472/273 (e) and threonine 308 (f). IL-12-stimulated cells were compared against unstimulated (negative control), 10% FBS stimulated (positive control), and cells stimulated with IL-12 plus PI3K inhibitor LY294002 (IL12+LY), FBS plus PI3K inhibitor (LY294002), or IL-12 plus pAkt selective inhibitor (IL12+MK). Beta-actin and total Akt serve as loading controls. Results are representative of at least 3 biological replicates