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Membranes were blocked with 5% non-fat milk and incubated with mouse anti-5mC (#61480, Active motif) or anti-5hmC (#39999, Active motif) followed by a horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology)

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[29] [30] Vials should not be frozen, and any cloudy or particulate solution should be discarded

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In obesity, the spontaneous (197, 198) and stimulated (199) pulsatile patterns of GH secretion governed by the hypothalamus are blunted, and the accumulation of abdominal adiposity, particularly visceral adipose tissue mass, was found to be a stronger negative determinant of GH secretion than other factors including age, sex, or generalized obesity (200202)