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

. 2006 Dec 27;1(1):e128. doi: 10.1371/journal.pone.0000128

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

Okabe et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

PMC Copyright notice

FBXW8 ubiquitinates cyclin D1 in a Thr286 phosphorylation-dependent manner. (A) IP-IB analysis (left). Protein from exponentially growing HCT 116 cells was precipitated with antibodies to cyclin D1 or IgG. Immunoprecipitates were subjected to SDS-PAGE and sequentially blotted with cyclin D1, CDK4, SKP1, CUL1 and CUL7 antibodies. IB analysis with 5% of total cell lysates was provided as a control (right). (B) IB analysis following depletion of SKP1 expression for 48 hrs after treatment with SKP1 siRNA double-strand oligonucleotides in HCT 116 cells. Non-targeting siRNA (Control) and mock transfection (−) served as controls. (C) IP-IB analysis. Twenty-six F-box full-length encoding cDNAs were cloned into V5 or Flag epitope tag expression vectors. These V5 or Flag-tagged F-box protein DNA plasmids were transfected together with HA-tagged cyclin D1 (HA-Cyc D1) and CDK4 expression vectors into T98G cells. Cells were collected 24 hrs later. Samples were precipitated with a HA epitope tag antibody. Immunoprecipitates were subjected to SDS-PAGE and subsequently stained with V5 or Flag (F-box proteins), HA (Cyc D1) antibodies. IB analysis with 10% of total cell lysates was provided (bottom). (D) IP-IB analysis (top). V5-tagged F-box protein DNA plasmids were transiently transfected together with either HA-tagged cyclin D1 (Cyc D1) wild type (WT) or T286A mutant, and CDK4 expression vectors in T98G cells respectively. Samples were precipitated with a HA epitope-tag antibody. Immunoprecipitates were subjected to SDS-PAGE and subsequently blotted with V5 (F-box proteins), HA (Cyc D1), and Thr286 phosphorylated cyclin D1 (pCyc D1 Thr286) antibodies. IB analysis with 10% of total cell lysates was provided for comparison (bottom). (E) In vitro ubiquitination assay. In vitro translated F-box proteins with recombinant GST-full-length cyclin D1 (Cyc D1) wild type, HeLa cell extracts Fraction II with ATP, Ubiquitin and ERK2, and in vitro-translated either SKP1, RBX1 and CUL1, or SKP1, RBX1 and CUL7 proteins were incubated at 30°C for 2 hrs. Samples were separated by SDS-PAGE and immunoblotted with a cyclin D1 antibody. (F) In vitro polyubiquitination of cyclin D1 through the SCF-like (SCFL) complex FBXW8 (SKP1-CUL7-FBXW8-RBX1/SCFL^FBXW8). WT or T286A GST- Cyc D1 was incubated in the presence of purified ERK2 (lanes 1, and 3) or its absence (lane 2) at 30°C for 2 hrs. Samples were separated by SDS-PAGE and immunoblotted with a cyclin D1 antibody. Asterisk indicates non-specific bands. (G) Reconstitution of polyubiquitination of cyclin D1 through SCFL^FBXW8 in vitro using purified E1 and E2. GST-WT Cyc D1 was incubated with recombinant SCFL^FBXW8 in the presence or absence of E1 and E2/UbcH5C. Samples were separated by SDS-PAGE and immunoblotted with a cyclin D1 antibody.