Chemically 'poisoned' protein acts as a molecular switch to spur cancer formation NatureComms
, and procedures were performed under dark conditions. Fresh SNOC or GSNO was diluted in 10 mM HEPES-NaOH . HEK293T cells were pretreated with the compound DBIC, DBIC-neg, or DBIC-der1 for 1 h prior to stimulation with NO donors. After washing the cells with phosphate-buffered saline , cell lysates were prepared in HEN buffer [250 mM HEPES-NaOH , 1 mM EDTA, 0.1 mM neocuproine] supplemented with 1% Triton X-100 and protease inhibitor cocktail .
5′-GGC GCT TAC CTT CTC CTA TGG-3′ and 5′-AAG CCC ATT CAC CTC AGC CC-3′; human CA9 5′-GAA ATC GCT GAG GAA GGC TC-3′ and 5′-CGG TGT AGT CAG AGA CCC CT-3′; human EGR1 5′-AGC ACC TGA CCG CAG AGT C-3′ and 5′-ACT GAC CAA GCT GAA GAG GG-3′; human GPR137C 5′-GGT CCT CTT TCT GTG GGA ACA-3′ and 5′-GCC AGC AGG TGC CAA ATT C-3′. The 2relative quantification method, using ACTB for normalization, was used to estimate the target gene expression.
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