Fig. 5.

Stimulation of CREB DNA binding activity is impaired in SH-SY5Y cells overexpressing GSK3β. (a) Cell lysates from wildtype SH-SY5Y cells (wt), vector-transfected SH-SY5Y cells (Vec), and four cell lines stably overexpressing HA-GSK3β (1, 3, 7, and 9) were immunoblotted for GSK3β. (b) Serum-starved (24 h) wild-type SH-SY5Y cells and GSK3β-overexpressing cells were treated without (basal) or with EGF (50 ng/mL; 1 h) or IGF-1 (50 ng/mL; 1 h) and lysates were immunoblotted for phospho-serine-473-Akt. (c) Serum-starved (24 h) wild-type SH-SY5Y cells and GSK3β-overexpressing cells were treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or lithium (1 mm; 1 h) and lysates were immunoblotted for phospho-serine-133-CREB. (d) The association of phospho-serine-133-CREB with CBP was measured by immunoprecipitating CBP from serum-starved (24 h) GSK3β-overexpressing SH-SY5Y cells treated without or with lithium (20 mm; 30 min), EGF (50 ng/mL; 30 min), or IGF-1 (50 ng/mL; 30 min) and immunoblotting phospho-serine-133-CREB, as described in the Materials and methods. (e) A representative CREB EMSA is shown obtained with GSK3β-overexpressing cells and quantitative data is shown of CREB DNA binding activity in nuclear extracts prepared from wild-type and GSK3β-overexpressing cells (cell lines 1, 3, 7, and 9) incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or forskolin (Fsk; 10µm; 1 h). Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 4–6. (f) EGR-1 DNA binding activity was measured by EMSA in nuclear extracts prepared from wild-type and GSK3β-overexpressing cells incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h) or IGF-1 (50 ng/mL; 1 h). ns signifies non-specific binding.