Determine whether MEK inhibition with trametinib alters the physical association between BRAF and MEK1 in BRAF-V600E A375 melanoma cells using endogenous co-immunoprecipitation and quantitative immunoblot detection of the co-precipitated partner.
Analyze the effects of oxidative stress on cellular damage pathways associated with tumor development.
Quantify EGF-induced phospho-ERK1/2 (Thr202/Tyr204) at single-cell resolution in MDA-MB-231 breast cancer cells by intracellular phospho-flow cytometry, resolving the kinetics and cell-to-cell heterogeneity of MAPK pathway activation.
Measure the catalytic activity of endogenous c-SRC kinase immunoprecipitated from HCT116 colorectal cancer cells before and after dasatinib treatment using a peptide-substrate phosphorylation assay, to confirm cellular target engagement of the SRC/ABL inhibitor.
Quantify the dose-dependent suppression of AKT Ser473 phosphorylation by the pan-PI3K inhibitor GDC-0941 (pictilisib) in PTEN-null PC-3 cells to determine the cellular IC50 for pathway inhibition.
Generate clonal RAF1 (c-RAF) knockout NCI-H358 KRAS-G12C lung adenocarcinoma cells by CRISPR-Cas9 ribonucleoprotein electroporation and validate loss of function through phospho-MEK1/2 immunoblot to assess RAF1 contribution to mutant-KRAS downstream signaling.