Fig. 6. Cell biology of ERK inhibitor activity in tumor cells. Western blot analysis to determine phosphorylation status of ERK1/2 and RSK in A549, DM122, and H82 cells treated with VX-11e and BVD-523. Cells were treated with each compound at respective concentrations for 4 hours in serum free media. Samples were analyzed with antibodies against p-ERK (Thr202/Tyr204) and p-RSK (Thr359/Ser363). (A, B) VX-11e and BVD-523 showed inhibition of phosphorylation for p-RSK at 0.5 μM and 2 μM. A549 and DM122 showed a slight increase in phosphorylation for ERK1/2 when treated with VX-11e and BVD-523. In contrast, H82 showed a massive enhancement in ERK1/2 phosphorylation upon compound treatment. (C) MS2 fragmentation spectra of RSK active-site peptides from quantitative chemoproteomic analysis. (D) SILAC analyses confirmed that VX-11e and BVD-523 are not inhibitors of endogenous RSK in A549 proteomes. Sensitivity of RSK peptide to ATP competition confirmed active site-dependent probe labeling in our chemoproteomic studies. RSK peptide used for analysis is shared between RSK1, RSK2, and RSK3.