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Treat cells by adding fresh media containing regulator for desired time. Wrap in plastic wrap and expose to x-ray film. Wash three times for 5 min each with 15 ml of TBST. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube.

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Sonicate for 10—15 sec to sie cell lysis and shear DNA to reduce sample viscosity. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Proceed with detection Section D.

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