Fig. 4.

Effect of silencing Sp4, depolarizing stimulation, and Sp4 overexpressing. (A– B) Western blots revealed a down-regulation of Sp4, GABA_A α1, and GABA_A α2, but not GABA_A α3 protein levels in Sp4 shRNA-transfected primary neurons. β-actin served as a loading control (N = 3). Using scrambled vector transfection as a control, primary neurons exposed to KCl up-regulated their mRNA levels for Sp4, Gabra1, Gabra2, and Gabra3 (* P < 0.05 when compared with controls; N = 3). When transfected with Sp4 shRNA, transcript levels of Sp4, Gabra1, and Gabra2 were significantly down-regulated, but not that of Gabra3 (* P < 0.05; ** P < 0.01; X = non-significant when compared to controls; N = 3). In the presence of Sp4 shRNA, KCl was no longer able to up-regulate mRNA levels of Sp4, Gabra1, and Gabra2 to that of control + KCl, though it was still able to do so for Gaba3 (## P < 0.01; ### P < 0.001; X = non-significant when compared to KCl alone). Transfection of neurons with Sp4 shRNA followed by Sp4 over-expression rescued their Sp4, Gabra1, and Gabra2 transcripts from being down-regulated by Sp4 shRNA, but this treatment had no effect on Gaba3 (⁺ P < 0.05; X = non-significant when compared to shRNA alone; N = 3). Neurons transfected with Sp4 shRNA followed by Sp4 over-expression as well as KCl treatment showed a level of Sp4, Gabra1, and Gabra2 transcripts that was significantly higher than that of shRNA + KCl, although it had no effect on that of Gabra3 (◇◇ = P < 0.01; ◇◇◇ = P < 0.001; X = non-significant when compared to shRNA + KCl; N = 3). All four transcript levels were not significantly different from those of control + KCl.