Figure 5.

IGF-1R is required for GABA_B receptor-mediated neuroprotection. A, Time course of IGF-1 (10 ng/ml)-induced IGF-1R and Akt phosphorylation in CGNs. B, Effect of pretreatment with AG1024 at 0.1 μm for 60 min on Akt phosphorylation, followed by induction by baclofen (100 μm) or CGP7930 (50 μm) for 10 min. To quantify pAkt or pIGF-1R, immunoblots from separate experiments (mean ± SEM; n = 3–5) were analyzed. For all results, *p < 0.05, **p < 0.01, and ***p < 0.001, versus the basal levels, and ⁺⁺⁺p < 0.001, versus treated with baclofen or CGP7930 in the absence of AG1024. C, Effect of AG1024 (0.1 μm) on apoptotic CGNs after baclofen (100 μm) stimulation. For all TUNEL assay (left) or Hoechst 33258 staining (right), cells were treated with drugs in K5 media for 18–20 h before fixing and staining. ***p < 0.001; ns, no significance, versus K5 conditions. The blots shown are representative of three separate experiments. D, Effect of AG1024 (0.1 μm) on caspase-3 activity in CGNs induced by GABA (100 μm), baclofen (100 μm), or CGP7930 (50 μm). The blots shown are representative of three separate experiments. For caspase-3 activation, cells were treated with drugs in K5 media for 2–12 h. E, Effect of shRNA of IGF-1R (IGF-R3467) on Akt phosphorylation induced by baclofen (100 μm) for 5 min in MEF cells cotransfected GABA_B1 and GABA_B2. Expression of IGF-R3467 decreased MEF cells endogenous IGF-1R expression but not Src kinase expression (left). The blots shown are representative of three separate experiments.