Figure 1: Ankyrin-G and the IKK-complex accumulate in synaptosomal and lipid fractions in the adult neocortex and co-immunoprecipitate.

(A) Fresh adult mouse neocortex tissue was isolated and separated using gradient centrifugation. Proteins were extracted from cellular fractions and analyzed by Western blotting. Note the co-localization of pan-IKKα/β, NEMO and p65/NF-κB immunoreactivities to the ankyrin-G (clone 463, SCBT) positive lipid fraction. (B) Optical densities (OD) of protein immunoreactivity from all fractions were determined and mapped as percentage of total OD of all fractions in bar graphs. (C) Ankyrin-G and the IKK-complex co-immunoprecipitate from adult mouse cortical lysates. Mouse cortices were homogenized and minced in Na-Sucrose buffer. Equivalent amounts were used for immunoprecipitation (IP) using anti-ankyrin-G, control anti-IgG, anti-MAP2 or anti-α-Tubulin antisera, followed by immuno-blotting (IB) using anti-ankyrin-G (463), anti-pan-IKKα/β (H-470) and anti-MAP2 antibodies. Full-blot views including spliced supernatant control bands in Suppl. Fig. 1A. (D) For the reverse experiment, cortical tissue was processed and immunoprecipitated using a rabbit polyclonal antibody to IKKα/β (H-470, SCBT), a rabbit polyclonal anti-IgG antiserum served as control. Rabbit-polyclonal antibodies against ankyrin-G (S.E. Lux) and an alternative commercial antibody raised against ankyrin-G (H-215) were applied to additional samples. Following electrophoresis and immunoblotting, anti-ankyrin-G (S.E. Lux) and anti-pan-IKKα/β (H-470) antibodies were applied in Western-blotting. Full-blot views included in Suppl. Fig. 1B. (E) The relative OD of anti-IKKα/β immunoreactivity of all three Ankyrin-G IP’s (Fig. 1C,D and Suppl. Fig. 1C) was plotted against respective IgG-control IPs. (F) Anti-GFP antibodies pulled Flag-tagged IKKβ down from ankG-eGFP co-transfected, but not eGFP co-transfected cells. AnkyrinG-eGFP pull-down experiments were conducted following a 2-day transfection of SH-SY5Y cells with ankG-eGFP and IKKβ-FLAG vectors. Tag-specific antibodies were used to detect the proteins following Western-blotting procedure. (G) Quantification of anti-FLAG immunoreactivity (ODs representing IKK-β) depicted in (F) and three additional, similarly conducted pull-down experiments was determined.