Figure 4

IGF1 induces greater levels of phospho-S6 kinase in KO GNPs compared to WT cells. (A) Untreated cerebellar lysates from freshly dissected 7-day-old (P7) WT and KO mice demonstrate identical baseline levels of both P-Akt and P-ERK. (B) The levels of P-Akt protein induced by a 30-min pulse of IGF1 was quantified in WT and KO GNPs by Western blot with densitometric analysis, and no significant differences were found between genotypes (C = control; I = IGF1). (C) IGF1 induced robust, but similar increases in P-Akt in both WT and KO GNPs after a 15-min pulse. This effect was attenuated at 4 h and 8 h, and no activity was found at 24 h (data not shown). P-ERK, on the other hand, appeared constitutively activated in these culture conditions, with no change in levels in either genotype following IGF1 treatment. (D) Phospho-S6 kinase, which is downstream of Akt, was markedly upregulated in KO GNPs pulsed 30 min with IGF1 compared to untreated GNPs. In contrast, IGF1 failed to upregulate phospho-S6 kinase in WT GNPs (though it robustly increased P-Akt), suggesting En2 may be an important negative regulator of the S6 kinase pathway. Total S6 kinase protein levels were not different between genotypes (not shown). Densitometry quantification in B and D is expressed in arbitrary units as percent control ± SEM. n = 3 experiments per genotype (3 animals per experiment, per genotype); #, Significance compared to genotype control, p ≤ 0.05; **, significance compared across genotypes, p ≤ 0.01.