Figure 3: Proximity-ligation assays (PLA) indicate molecular proximity between IKKα/β and ankyrin-G.
From: NF-κB regulates neuronal ankyrin-G via a negative feedback loop
(A) Principle of proximity-ligation assays to detect proximity of the IKK-complex and ankyrin-G. Oligonucleotide-labeled antibodies hybridize and are amplified via rolling-circle amplification when in molecular proximity (
40 nm). (B–E) Panels depicts mature cultured cortical neurons transfected with eGFP, EB1-eGFP, as negative control or ankG-eGFP with or without IKKβ expression plasmids. PLA between anti-EB1-anti-panIKKα/β as negative control, or anti-ankyrin-G and anti-panIKKα/β epitopes, was conducted using anti-mouse and anti-rabbit specific antibodies. Arrows depict PLA-reactivity in the axon. The experiment was repeated under similar conditions with comparable results. (G) Somatic and proximal PLA-spots were counted from 3-183 transfected and un-transfected cells pooled from 1–2 experiments. Kruskal-Wallis, Dunn’s post-hoc test. Scale bars 10 μm.
