Crizotinib and PF-06463922 Inhibit ALK Signaling

PC12 cells were grown and transfected with various ALK constructs as described (Chand et al., 2013 (link)). 48 h after transfection, cells were treated with different concentrations of crizotinib (from 0 to 5000 nM) or PF-06463922 (from 0 to 500 nM) for 2 h. Cells were then washed twice with PBS and harvested as described (Schonherr et al., 2012 (link), 2011 (link)). Samples were boiled in SDS sample buffer and analyzed by immunoblotting. Intensity of pALK (Y1604) and total ALK bands was quantified with Image Studio Lite 3.1 (LI-COR). Data were normalized to the 0 nM inhibitor samples. IC₅₀ values were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0.