Skip to main content
. 2013 Mar 11;32(46):5359–5368. doi: 10.1038/onc.2013.40

Figure 3.

Figure 3

Mer stimulation in AML cells activates oncogenic signaling pathways. (a) Immunoblot analysis of Kasumi-1 and Nomo-1 cell lines confirmed they were positive for Mer (∼180 kD), but negative for other Tyro-3/Axl/Mer family tyrosine kinases Axl (140 kD) and Tyro-3 (140 kD). (b) Kasumi-1 and Nomo-1 cells were incubated in serum-free RPMI medium then treated with 200 nM rhGas6 (Gas6 treated) or buffer (Untreated). Immunoprecipitate analysis demonstrating phosphorylation of Mer after 200 nM Gas6 treatment (+) compared with treatment with buffer (−). Total Mer was used as a loading control. (c) Similarly treated diluted lysates were incubated with human phospho-kinase array membranes and bound phospho-proteins were detected according to kit instructions. Each membrane contains positive control (P) antibodies spotted in duplicate. Proteins which demonstrated a 1.5-fold or greater increase in phosphorylation after stimulation with Gas6 (Gas6 treated) relative to buffer treated (Untreated) are marked by numbers between duplicate spots, which correlate with the identification numbers shown in (d). (d) Levels of the indicated phospho-proteins were quantified after normalizing pixel density of each positive control to 100. Untreated samples are denoted in white, and Gas6 treated cells are denoted in gray hatched lines, showing the difference in relative phosphorylation between the two conditions. Each bar represents the mean±s.e. of duplicate spots. Increases were found in levels of phosphorylated MSK1 (S376/S360), AMPKα (T174), AKT (S473), mTOR (S2448), CREB (S133), SRC family kinases SRC (Y419), LYN (Y397), YES (Y426), FGR (Y412) and HCK (Y411) and STAT6 (Y641). (e) Kasumi-1 and Nomo-1 cells were incubated in serum-free media for 2–3 h followed by treatment with 200 nM rhGas6 (+) or buffer (−). Whole-cell lysates were subjected to immunoblot analysis with antibodies specific for the phosphorylated and total MSK1, p38, ERK1/2, STAT6, AKT, ATF1 and CREB proteins. Representative Nomo-1 immunoblot shown here. (f) Oncogenic downstream signaling pathways found to be activated in Mer-positive AML cells following stimulation with Gas6.