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. 2017 Mar 1;130(5):975–988. doi: 10.1242/jcs.197343

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© 2017. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 5. — IKKα sulfenylation promotes keratinocyte migration. (A) Upper blot: DYn-2 probe-loaded keratinocytes were biotinylated using ‘click chemistry’ with a biotinylated azide probe. Sulfenylated proteins were probed with streptavidin (Strep)–HRP (upper panel). IKKα is detected in all fractions (with or without DYn-2 and H₂O₂). Lower blot: H₂O₂ oxidizes IKKα, assessed after purification of the biotinylated fraction by streptavidin pull-down and probing with anti-IKKα antibody. GAPDH serves as positive control for successful pull down of oxidized/sulfenylated proteins given its redox function (Truong, 2012a). IP, immunoprecipitation; WB, western blot. Values under the blots depict fold-changes from baseline controls. (B) Human (Hu) and zebrafish (z) IKKα kinase domain sequence comparison shows high conservation (∼70%), including zebrafish residue cysteine 179 (arrow). Red, conserved residues; blue, non-conserved residues. (C) IKKα construct scheme for HEK001 in vitro transfection studies are shown to the right of panel D. Full-Length (FL) Ikkα–tdTomato in cytoplasmic perinuclear regions with and without H₂O₂ (white arrowheads). Large arrowheads indicate cells magnified in the insets. DPI treatment (10 µM) results in punctate localization within subregions of the nucleus. Scale bar: 100 µm. (D,E) Point mutant (C179A) shows nuclear localization without H₂O₂ (D) and with H₂O₂ (0.1 µM) (E). Scale bar: 100 µm. Arrowheads, indicate transfected cells; large arrowheads indicated cell magnified in the insets. (F) Cell movement and directionality (arrows) tracked over time. Control (untreated) cells migrate slightly faster than cells treated with high H₂O₂ (100 µM) concentrations, following DPI treatment and when transfected with C179A-Ikk. Transfection with FL-Ikk and treatment with low H₂O₂ (0.1 µM) concentrations promotes migration. Scale bar: 40 µm. Ave, average, mean; LZ, leucine zipper; HLH, helix-loop-helix; NBD, Nemo-binding domain. Data are mean±s.d. ANOVA test and Tukey's multiple comparisons test were used for statistical analyses.