Figure 1.

EP009 is a selective inhibitor of JAK3 with anti-cancer activity. (a) Chemical structure of EP009 (M.W. 224.34). (b) In vitro autokinase analysis of immunopurified JAK3 treated with vehicle (DMSO; lanes a and b) or ascending concentrations of EP009 (0–10 μM; lanes c–f). The reactions were incubated in the absence (lane a) or presence (lanes b–f) of 1 μM ATP prior to separation by 7.5% SDS-PAGE and subsequently Western blotted (WB) with anti-phosphotyrosine (α-pY). The blot was stripped and reprobed with α-JAK3 to confirm equivalent loading. (c) Kit225 (upper panel) or BaF/3 (lower panel) cells cultured in the presence of IL-2 or IL-3, respectively, were treated with vehicle (PBS; lane a) or increasing amounts of EP009 (0–50 μM; lanes b–h) for 12 hours. Cells were then lysed, clarified, and immunoprecipitated (IP) with anti-JAK3 (α-JAK3) or anti-JAK2 (α-JAK2), and subjected to WB analysis with α-pY. Blots were stripped and reprobed with corresponding antibody to verify equivalent protein loading. (d–h) The indicated cells were cultured with increasing amounts of EP009 (0–10 μM) for 72 hours and cell viability measured with the MTS tetrazolium salt assay. Values represent mean absorbance (OD₄₉₀-OD₆₅₀ nm) normalized to vehicle (PBS) treated control cells, while error bars represent the standard deviation (n = 3). (g, h) Additionally, total cell lysates were separated by 7.5% SDS-PAGE and subjected to WB analysis with α-JAK3. Blots were then stripped and reprobed with α-GAPDH to verify equivalent protein loading. Representative data from three independent experiments are shown.