Supplementary Figure 12: Characterization of AAV-HA-caIKK-β-p2A-eYFP viral vector, and effect of AAV-HA-caIKK-eYFP on HDAC1
From: Antipsychotic-induced Hdac2 transcription via NF-κB leads to synaptic and cognitive side effects
(a) Schematic representation of the recombinant AAV vector used to over-express both HA-tagged constitutively active IκB-kinase (IKK-β-S177E-S181E; HA-caIKK-β) and eYFP under the CaMKIIα promoter in mouse frontal cortex pyramidal neurons. The arrowhead indicates p2A cleavage site. (b) AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP was injected into the frontal cortex, and HA expression was measured by Western blotting at the indicated time after surgery (HA-caIKK-β: ∼ 85 kDa). (c) Representative immunohistochemical images of mouse frontal cortex injected with the AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP construct. Note that signals of HA-caIKK-β (red) and eYFP (green) do not overlap, indicating high cleavage efficiency of the p2A site. (d) Neuro2A cells were transfected with either AAV-CaMKIIα::eYFP (AAV-eYFP) or AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP (AAV-HA-caIKK-β-p2A-eYFP). Western blot with anti-HA antibody showed the expected molecular weight of HA-caIKK-β in cells transfected with AAV-HA-caIKK-β-p2A-eYFP, indicating high cleavage efficacy of the p2A peptide. (e) Neuro2A cells were transfected with the AAV-CaMKIIα::HA-caIKK-β-p2A-eYFP construct. Red signal (HA-caIKK-β) that does not overlap with green signal (eYFP) indicates high cleavage efficacy of the p2A peptide. (f,g) AAV-mediated over-expression of HA-caIKK-β-eYFP does not affect HDAC1 in eYFP-positive frontal cortex neurons. Representative immunohistochemical images (f). Quantitative assessment (HDAC1, n = 13-21 cells from 4 mice per experimental condition, g). Two-way ANOVA with Bonferroni’s post-hoc test (g, P = 0.84, F1,70 = 0.03). Nuclei were stained in blue with DAPI (b,e,f). Scale bars, 10 μm (b), 20 μm (f).
