Fig 2: PKA-Notch pathway is involved in the NICD/RBPJk-dependent transcription during C6 astrocytic differentiation(A) C6 cells were transfected with 0.5 µg of the indicated reporter vectors for recording NICD/RBPJk-dependent transcription (4xwtCBF1Luc, wild-type/4xmtCBF1Luc, mutant version; schematic representations are shown in the upper part of the Figure); 24 h after transfection, cultures were treated with dbcAMP for another 24 h; when indicated, inhibitors were added as in Figure 1. Co-transfection assays were performed with 1.25 µg of 4xwtCBF1Luc or 4xmtCBF1Luc and 3.75 µg of NICD expressing vector (pFCDN1 and the pEF1a-BOS empty vector used as controls). (B) At 24 h after transfection with 0.5 µg of 4xwtCBF1Luc vector, cultures were treated with dbcAMP (750 µM) or forskolin (FSK, PKA inductor, indicated concentrations) for another 24 h; inhibitors were added 30 min before dbcAMP or FSK. (C) C6 cells were transfected with 2 µg of pCRE-CAT reporter construct, and 24 h after transfection treated with dbcAMP or FSK (as indicated) for another 24 h. In all cases, data are shown as relative to the activities in the non-treated (NT) control or the control co-transfected with the empty vector. (D) Total RNA was obtained from C6 cells treated as indicated and processed for qRT-PCR to amplify a 173 bp fragment from PEN-2 mRNA (nucleotides 143–315), using GAPDH levels as reference. Data are plotted relative to values recorded in non-treated (NT) and DMSO controls. (E) Approximately 100 µg of total protein extract from C6 cells treated with dbcAMP (750 µM, 2 days), were used for Western blot analysis with anti-PEN-2 and anti-GAPDH antibodies. Molecular masses are depicted on the right side. In all cases, data are plotted as means±S.E.M. from at least three independent experiments; statistical analysis was performed using a non-parametric one-way ANOVA (Kurskal–Wallis test) and Dunn's post-hoc test (*P<0.05; **P< 0.01; ***P<0.001).