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. 2009 Jan 19;206(1):221–232. doi: 10.1084/jem.20082044

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© 2009 Massoumi et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — CYLD regulates N-cadherin and Cyclin D1 expression via BCL-3 in melanoma. (A) Western blot analysis revealing CYLD expression in NHEMs and Mel Im and Mel Juso cells stably transduced with CYLD, but not in melanoma cells stably transduced with GFP. Actin labeling is used as loading control. (B) Western blot detecting Cyclin D1 expression in Mel Im cells stably transduced with CYLD, but not in nontransduced control cells or cells stably transduced with GFP. Tubulin labeling is used as loading control. (C) Effect of transfection of a CYLD expression vector carrying wild-type (CYLD) or mutated CYLD (CYLD C/S) on Cyclin D1 promoter activity in Mel Im cells. Data are given as mean ± SEM. *, P < 0.05 versus pcDNA3 empty vector used as control. (D) Lysates from Mel Im cells (Control) and CYLD, mutant CYLD (CYLD C/S), or GFP stably expressing Mel Im cells were examined by ChIP assay using specific polyclonal antibodies against BCL-3, p50, or p52, and PCR primer pairs corresponding to the promoter of the CyclinD1 or N-cadherin gene to analyze recruitment of BCL-3 to the Cyclin D1 and N-cadherin promoter. BCL-3, p50, and p52 immunoprecipitation (IP) using polyclonal antibodies as indicated. IgG, negative control rabbit IgG (DAKO); Input, 10% of the cell lysate used for the IP is shown. (E) Effect of transduction of a CYLD expression vector carrying wild-type (CYLD) or a catalytic inactive mutant of CYLD (CYLD C/S) on N-cadherin promoter activity in Mel Im cells. Data are given as mean ± SEM. *, P < 0.05 versus pcDNA3 empty vector used as control. (F) Western blot analysis of CYLD, Snail1, Snail2, Twist, E-cadherin, N-cadherin, and Actin expression in nontransduced Mel Im cells (Control) or Mel Im cells transduced with GFP or CYLD. (G) Western blot analysis of BCL-3, Cyclin D1, N-cadherin, Snail1, and Actin expression in nontransduced Mel Im and Mel Juso cells (Control), cells transduced with the BCL-3 siRNA nucleotides (siRNA BCL-3), or scrambled siRNA control (siRNA control). (H) Western blot analysis of Snail1, CYLD, (nuclear) BCL-3, and Actin expression in nontransduced Mel Im cells (Control), and cells transduced with Snail1 siRNA nucleotides (siRNA Snail1) or scrambled siRNA control (siRNA control). Experiments in A–H were repeated at least three times.