TY - CHAP
T1 - Rat Brain/Hep G2 Glucose Transporter Gene Expression in Brain
AU - Raizada, Mohan K.
AU - Rydzewski, Bartosz Z.
AU - Werner, Haim
AU - Masters, Brian A.
AU - Roberts, Charles T.
AU - Leroith, Derek
PY - 1992/1/1
Y1 - 1992/1/1
N2 - This chapter presents a methodology to quantitate Glut-1 mRNA and protein levels in the brain and in neuronal and astroglial cells in primary culture. Measurement of Glut-1 mRNA levels include isolation of total RNA, cDNA probe preparation, Northern blotting, and hybridization. In a study described in the chapter, a single ∼2.8-kb band that hybridized with Glut-1 cDNA probe was observed at each stage of brain development. The steady state levels of Glut-1 mRNA were relatively low at embryonic day 20 and at early postnatal stages. These levels increased around weaning time (postnatal day 21) and reached maximal values in the adult brain. Using scanning densitometry, it was found that Glut-1 mRNA levels in the adult brain were three- to fourfold higher than at birth. The chapter also discusses the measurement of Glut-1 protein by western blotting. It presents an autoradiogram of a representative experiment where Glut-1 protein levels have been measured in astroglial cells in response to the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA). TPA stimulation of [3H]deoxy-D-glucose uptake in astrocytes is associated with an increase in the amount of Glut-1 protein.
AB - This chapter presents a methodology to quantitate Glut-1 mRNA and protein levels in the brain and in neuronal and astroglial cells in primary culture. Measurement of Glut-1 mRNA levels include isolation of total RNA, cDNA probe preparation, Northern blotting, and hybridization. In a study described in the chapter, a single ∼2.8-kb band that hybridized with Glut-1 cDNA probe was observed at each stage of brain development. The steady state levels of Glut-1 mRNA were relatively low at embryonic day 20 and at early postnatal stages. These levels increased around weaning time (postnatal day 21) and reached maximal values in the adult brain. Using scanning densitometry, it was found that Glut-1 mRNA levels in the adult brain were three- to fourfold higher than at birth. The chapter also discusses the measurement of Glut-1 protein by western blotting. It presents an autoradiogram of a representative experiment where Glut-1 protein levels have been measured in astroglial cells in response to the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA). TPA stimulation of [3H]deoxy-D-glucose uptake in astrocytes is associated with an increase in the amount of Glut-1 protein.
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U2 - 10.1016/B978-0-12-185267-2.50011-8
DO - 10.1016/B978-0-12-185267-2.50011-8
M3 - Chapter
AN - SCOPUS:0009687277
T3 - Methods in Neurosciences
SP - 79
EP - 88
BT - Methods in Neurosciences
ER -