Figure 2: The IKK-complex co-localizes with ankyrin-G to the axon initial segment.

(A–D) Cortical neurons were cultured until maturity in vitro and were cytosol-extracted for the visualization of cytoskeletal-associated proteins. They were incubated for 5 minutes in 1% (m/V) Triton-X-100 (A–C) or 0.5% saponin (D) in 10 mM Na₃PO₄-buffer (pH 7.4), 1 mM MgCl₂, 3 mM CaCl₂, 150 mM NaCl. Extractions were performed at 4 °C, prior to fixation and immunostaining. Anti-IKKγ/NEMO immunoreactivity is highlighted in red (A) or green (B), the AIS-specific marker⁴⁴ antibody 14D4 (green,A) or anti-AnkG (red, B) were used to identify the AIS (green). (C,D) Extractions were similarly performed followed by immunolabeling using pan-IKKα/β - specific antibodies (red, rabbit polyclonal, H-470, SCBT) with the AIS labeled by anti-ankyrinG in green. Color overlays are depicted following background substraction and sharpening, arrows highlight AISs, scale bars, 10 μm. (E) IKKβ-Flag and eGFP vectors were transfected into mouse cortical neurons on DIV13 using Ca-precipitation. 2 days following transfection, neurons were fixed and immunolabeled using a rabbit polyclonal anti-Flag-specific antibody (Sigma, red), as well as a mouse monoclonal ankyrin-G-specific antibody (Zymed). Note the Flag-tag-specific immunoreactivity within the ankyrin-G positive AIS stretch (arrow, yellow). (F) IKKβ-Flag and ankG-eGFP vectors were transfected into mouse cortical neurons as above. 2 days following transfection neurons were cytosol-extracted for visualization of cytoskeletal-associated proteins by incubation in 1% (m/V) Triton-X-100 buffer for 5 minutes. Extractions were performed using pre-cooled solutions and performed at 4 °C, prior to fixation and immunostaining. Note that overfixation often obliterates AIS-immunoreactivity. The inset shows a magnified view of the anti-Flag positive AIS. Note that ankG-eGFP and anti-Flag reactivity remained associated with the non-soluble cytoskeleton in both, in the AIS (arrow), but also in spots within the soma following Triton-X100 extraction. The ankyrin-G antibody showed a notably stronger immunolabeling in the ankG-eGFP transfected cell than in the surrounding untransfected control cells. Scale bars, 10 μm.