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Transient transfection of the PX459-sgRNA into the target cancer cell lines was performed using the Lipofectamine 3000 Transfection Reagent (Invitrogen) in 6-well plates (Corning Falcon) followed by puromycin selection (Gibco, 15 g/ml) for 3 days
Urea (8 M) was added to the precipitate to dissolve the RIPA-insoluble protein components
This inevitably results in low IGF-I and high GH that may stray into the acromegaly range (132, 133) and this catabolic state is comparable to prolonged fasting and malnutrition states ( Figure 1E )
To address the role of the activator candidate for glutaredoxin catalysis, we replaced Lys105 of ScGrx7 by site-directed mutagenesis
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