Evaluating Signaling Pathway Responses

Cells were treated with WX-554 and WX-037 at 1 or 10 times the half maximal growth inhibitory concentration (GI₅₀) in DMSO, or 0.5% (v/v) DMSO alone, for 24 h. Western blots were prepared, probed with phospho-4EBP1 (Thr37/46) (#2855), phospho-p44/42 MAPK (Thr202/Tyr204) (#4370), phospho-AKT (Ser473) (#4060) or phospho-S6 ribosomal protein (Ser235/236) (#4858) monoclonal antibodies obtained from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hertfordshire, UK) and developed as described previously [23 (link)]. Blots were then stripped (100 mM 2-mercaptoethanol, 2% (w/v) SDS and 62.5 mM Tris pH6.8 at 55 °C for 30 min) and re-probed with the respective total monoclonal antibody (4EBP1 (53H11) (#9644), p44/42 MAPK (ERK1/2) (#4695), AKT (pan) (C67E7) (#4691) or S6 ribosomal protein (5g10) (#2217)) obtained from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hertfordshire, UK) and developed as described above.