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Lysates were boiled at 95 C for 5 min, 20 g of total protein were subjected to separation by a 10% SDS gel, transferred onto nitrocellulose membranes (Amersham Biosciences) and blocked with 5% milk powder in PBS Tween (0.1%) for 1 h

These effects develop gradually over weeks to months as weight normalizes and metabolic function improves

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