Figure 7.

PLC downstream effectors FAK1 but not Src kinase is involved in GABA_B receptor-mediated IGF-1R transactivation. A, Cells were pretreated with U73122 (5 μm) or U73343 (5 μm) for 60 min and induced by baclofen at 100 μm for 10 min. IGF-1R phosphorylation was measured by immunoblotting. B, PP2 (5 μm) was used to pretreat cells for 60 min or AG1024 (0.1 μm) before baclofen (100 μm) for 10 min. IGF-1R phosphorylation was detected by immunoblotting. C, Time course of baclofen (100 μm)-induced Src kinase phosphorylation in CGNs. D, Effect of pretreatment with U73122 (5 μm) or AG1024 (0.1 μm) on Src kinase phosphorylation induced by baclofen (100 μm) for 10 min. E, Effect of intracellular Ca²⁺ and FAK1 on baclofen-induced Akt phosphorylation for 10 min. Cells were pretreated with cell membrane-permeable Ca²⁺ chelator BAPTA-AM (10 μm) for 30 min and FAK1 inhibitors [FAK14 (50 μm) or PF573228 (10 μm)] for 10 min before the CGNs were stimulated with baclofen (100 μm) for 10 min, respectively. Akt phosphorylation was measured by immunoblotting. For all results, representative immunoblots are shown under the quantified data of pIGF-IR, pSrc, or pAkt analyzed from separate experiments (mean ± SEM; n = 3–5). *p < 0.05, **p < 0.01, and ***p < 0.001, versus the basal levels, and ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001, ns, no significance, versus treated with baclofen in the absence of inhibitors. F, MEF cells cotransfected with GABA_B1 and GABA_B2 were pretreated with control RNAi or RNAi of FAK1 for 48 h and then stimulated with baclofen for 5 min, respectively. IGF-1R and Akt phosphorylation were measured by immunoblotting. The blots shown are representative of three separate experiments.