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. 2012 Apr 18;32(16):5688–5703. doi: 10.1523/JNEUROSCI.0111-12.2012

Figure 3.

Figure 3.

Synapse formation is dynamically regulated by IKK/NF-κB signaling ex vivo. A, Coimmunolabeling of DIV21 hippocampal cultures from wild-type mice with IKK1/2, NEMO, p-IKK (all green), and the postsynaptic marker ProSAP1/Shank2 (red). Colocalization with ProSAP1/Shank2 is marked by filled arrowheads in case of IKK1/2 and NEMO and by framed arrowheads in case of p-IKK. B, IKK2-DN transgene expression in cultured neurons (DIV21) from control and IKK2nDN animals. Immunoblot from two independent hippocampal cultures is shown. Top panel, DIGNAM extracts, Bottom panel, RIPA extracts. RIPA extract indicates IKK2-DN localization in membrane-associated fractions. Loading controls, Erk2. C, Coimmunolabeling of DIV21 hippocampal neurons from control (Co) and IKK2nDN mice for VSV (green) and MAP2 (red). D, Quantification of excitatory synapse numbers at the indicated time points of hippocampal in vitro synaptogenesis from control and IKK2nDN mice (left panel) and representative images of secondary dendrites (15-μm-long segments) from control and IKK2nDN DIV17 hippocampal neurons coimmunolabeled either for Bassoon (presynaptic specializations, green-colored dot-like structures) and MAP2 (dendrite, red-filled structure) or Bassoon (green) and ProSAP1/Shank2 (postsynaptic specializations, red-colored dot-like structures) (right panel). E, Direct comparison of reduced synapse numbers from hippocampal cultures expressing a transdominant IκBα (ΔNIκBα) or IKK2-DN (IKK2nDN) protein. F, Representative images of secondary dendrites (15-μm-long segments) from control, IKK2nDN and DOX-treated IKK2nDN hippocampal neurons coimmunolabeled for Bassoon (green) and ProSAP1/Shank2 (red) (left panel). G, Quantification of synapse numbers from the conditions depicted in F. H, Quantification of synapse numbers from DIV16 IKK2nDN hippocampal neurons treated with DOX for the indicated time periods. G, H, RT-PCR analysis of mRNA from control (Co), untreated, and DOX-treated IKK2nDN hippocampal neurons (bottom panels). Duration of treatment was as indicated. IKK2-DN indicates transgene expression. Loading controls, Actin. I, Quantification of synapse numbers from DIV16 control hippocampal neurons after 24 h treatment with IKK2 inhibitor (BI 5700) at the indicated concentrations. A–I, Co, Control. All quantitative data are shown as mean ± SEM. All p values are derived from Student's unpaired, two-tailed t test. *p < 0.05; **p < 0.01; ***p < 0.001.