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Validation of PD98059 Inhibition on MAPK Activation in WT-Ras Transfected MCF-7 Cells

This study investigates the inhibitory effects of PD98059 on MAPK activation, as evidenced by the levels of phosphorylated ERK1 and ERK2 in MCF-7 cells. Cells were transiently transfected to express wild-type Ras and either stimulated with TNFα (50 ng/ml) or left untreated. PD98059 (50 μM) was added 2 hours prior to stimulation and maintained throughout the assay. Western blot analysis was conducted to assess ERK activation levels, confirming the efficacy of PD98059 as a MEK inhibitor in modulating ERK signaling in response to TNFα.

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Validation of PD98059 Inhibition on MAPK Activation in WT-Ras Transfected MCF-7 Cells

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  1. Additional File 4 Cells: WT-Ras 0 min 5 min 10 min Time of stimulation by TNFα: Phospho ERK1 (44 kDa) Phospho ERK2 (42 kDa) Total ERK1 (44 kDa) Total ERK2 (42 kDa) -- -- -- PD98059 PD98059 PD98059 • Additional File 4 • Validating the inhibitory functions of PD98059 on MAPK activation, indicated by levels of phosphorylated Erk • MCF-7 cells were transiently transfected to express WT-Ras and were not-stimulated or stimulated by TNF (50 ng/ml). This procedure was performed in the absence or presence of the MEK inhibitor PD98059 (50 μM), or its solubilizer (DMSO, at similar dilution). PD98059 was added to cell cultures 2 hr prior to stimulation of the cells by TNFα, and was present in culture throughout the duration of stimulation. Erk activation was determined by WB.

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