Modulating Protein Expression in Cells

To knock down expression, we used predesigned sets of 4 independent siRNA sequences of the target genes (siGENOME SMARTpool, Dharmacon, and Thermo Scientific, Pittsburgh, PA). To achieve overexpression of Chk1 and TAZ, we used pcDNA4-Chk1-Flag for Chk1 and pCMV5-TOPO-3Xflag-TAZ for TAZ (Addgene). Controls included cells that were mock transfected (no siRNA or DNA), those transfected with vector alone (for overexpression experiments), and those transfected with a nontargeting (scrambled) siRNA (for knockdown experiments). Cells were harvested, washed, and suspended (2 × 10⁶/100 μL) in Nucleofector V solution (Lonza Group, Walkersville, MD). siRNA (200 pmol/100 μL), DNA (3 μg/100 μL), or controls were added and electroporated using the U031 or U024 Nucleofector program (Lonza) as described previously [46 (link)]. ^WTBRAF overexpression was performed by Lipofectamine 3000 Transfection Kit (ThermoFisher Scientific, Grand Island, NY) according to the manufacturer's instruction. Overexpression or knockdown was confirmed using Western blot analysis as described above.