Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2015 Mar 15;29(6):672–685. doi: 10.1101/gad.254292.114

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2015 Yan et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

PMC Copyright notice

Figure 2. — JFK promotes ING4 degradation through an SCF-dependent pathway. (A) Wild-type JFK promotes ING4 degradation in breast cancer cell lines. MCF-7 and MDA-MB-231 cells were transfected with empty vector or Flag-tagged JFK or treated with control siRNA or JFK siRNA. Cellular extracts were prepared, and Western blotting was performed with the antibodies against the indicated proteins. (B) Wild-type JFK, but not JFK mutants, promotes ING4 degradation. MCF-7 cells were transfected with Flag-tagged JFK, JFKΔF-box, or JFKΔKelch. Cellular extracts were prepared, and Western blotting was performed with the antibodies against the indicated proteins. (C) JFK negatively affects the steady-state level of the ING4 protein. MCF-7 cells were transfected with increasing amounts of Flag-JFK. Forty-eight hours after transfection, cells were treated with DMSO or MG132 for 6 h before cells were collected for Western blotting analysis. (D) The destruction of ING4 by JFK is mediated by an SCF complex. MCF-7 cells were transfected with Flag-JFK and treated with siRNAs for Cul1, Skp1, or Cdc20 as indicated. Cellular proteins were extracted for Western blotting analysis with antibodies against the indicated proteins, or total RNAs were extracted and analyzed for ING4 expression by real-time RT–PCR. Bars represent the mean ± SD for triplicate experiments. (E) JFK negatively regulates ING4 half-life. MCF-7 cells were transfected with empty vector or Flag-JFK or treated with control siRNA or JFK siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL cycloheximide (CHX) for the indicated times in the presence or absence of MG132 before cellular proteins were extracted for Western blotting analysis. Quantitation was done by densitometry and expressed as signals of ING4/β-actin. Bars represent the mean ± SD for triplicate experiments. (F) JFK siRNA is specific. MCF-7 cells were transfected with vector or a JFK siRNA-1-resistant form (rJFK) together with control siRNA or JFK siRNA. Cellular proteins were extracted for Western blotting analysis with antibodies against the indicated proteins.