Figure 5.

Gadd45γ regulates MAPK signaling in mouse hippocampal cultures. A, Schematic representation of the experimental design. B, Schematic representation of Gadd45γ–shRNA2 viral construct. The viral vector contains a U6 promoter driving Gadd45γ-specific shRNA sequence (Gadd45γ–shRNA2) and a cytomegalovirus (CMV)/chicken β-actin (CBA) hybrid promoter driving GFP as an infection marker. Representative images of cultured hippocampal cells infected with this virus are shown. Scale bar, 40 μm. bGH polyA, Bovine growth hormone polyadenylation signal; ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. C, Schematic representation of the experimental design. D, qRT-PCR analysis of Gadd45α (n = 2 independent neuronal cultures), Gadd45β (n = 5 independent neuronal cultures), and Gadd45γ (n = 5 independent neuronal cultures) expression levels in hippocampal-cultured cells infected with control–shRNA or Gadd45γ–shRNA2 under basal conditions (left) or after 2 h bicuculline treatment (right). Expression levels were normalized to the corresponding uninfected controls in baseline conditions or upon bicuculline treatment (dashed line). E, Representative immunoblot scans using phosphospecific and total protein antibodies in hippocampal cultures infected with rAAV expressing control–shRNA, Gadd45β–shRNA, Gadd45γ–shRNA1, or Gadd45γ–shRNA2. Hippocampal cultures were harvested at baseline conditions or after 1 h of bicuculline treatment. F–H, Immunoblot quantification of p38 (n = 5–8 independent neuronal cultures; F), JNK (n = 5–8 independent neuronal cultures; G), and ERK (n = 4–7 independent neuronal cultures; H) presented in ratios of phosphorylated/total protein normalized to corresponding uninfected 1 h bicuculline condition (dashed lines). *p < 0.05, ***p < 0.001, ****p < 0.0001 by two-tailed unpaired Student's t test. ns, Not significant. Error bars represent SEM.