Fig. 1.
Impact of DEL-22379 and U0126 on ERK dimerization and signaling in thyroid tumor BRAF- and RAS-mutant cells. 8505c and OCUT2 cells carry BRAFV600E mutations and CAL62 and HTH83 cells carry K-, and H-RAS mutations, respectively. Western blot assays are representative from 3 independent experiments. A Native western blot assay of cells pre-treated for 30 min with 10 µM DEL-22379, 10 µM U0126 or vehicle before stimulation for 5 min with 100 ng/ml EGF. For ERK2 protein analysis, the upper band in the gels corresponds to the monomeric (m) form and the lower band to the dimeric (d) form. B Protein extracts from (A) were separated in a SDS-PAGE western blot assay. pT202/Y204ERK1/2 was detected to monitor ERK activation, pS380RSK was detected to monitor ERK cytoplasmic activity, pS218/S222MEK1/2 was detected to show upstream activation of the pathway and vinculin was used as a loading control. C mRNA levels of the ERK nuclear effectors FOS, JUN and EGR1 after 48 h in the presence of 10% FBS plus 1 µM DEL-22379, 10 µM U0126 or vehicle (DMSO), estimated by RT-qPCR. Results are expressed as mean (SD) of at least 3 independent experiments. Statistical significance of differences elicited by treatments compared with a vehicle was calculated using a two-tailed t-test: (*)p = 0.05–0.01, (**)p = 0.01–0.001. D Representative western blot of three independent experiments showing FOS and JUN protein levels after 48 h in the presence of 10% FBS plus DMSO (−), 1 µM DEL-22379 (D1), 5 µM DEL-22379 (D5) or 10 µM U0126 (U0). Vinculin is shown as a loading control