Journal: The Journal of Cell Biology

Article Title: Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

doi: 10.1083/jcb.200802076

Figure Lengend Snippet: Expression and targeted inactivation of Fbw7 isoforms. (A) Fbw7 isoform mRNA abundance in the indicated cell types. (B) 293A cells were cotransfected with vectors encoding FLAG-tagged Fbw7 (0.3 μg Fbw7α and 3 μg each of Fbw7β and Fbw7γ) and treated with cycloheximide (CHX) as indicated. The three isoforms are indicated. (C) Schematic demonstrating locations of inserted stop codons for each knockout genotype (see text). (D) Lysates were prepared from confluent p150 plates of Hct116 clones of the indicated genotypes and immunoprecipitated with polyclonal anti-Fbw7 antibody followed by immunoblotting with monoclonal anti-Fbw7 antibody. (E) Real-time PCR analysis of relative Fbw7 isoform mRNA expression in Hct116 Fbw7 knockout cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates).

Article Snippet: Hct116 (J. Simon, Fred Hutchinson Cancer Research Center, Seattle, WA), human foreskin fibroblasts (W. Carter, Fred Hutchinson Cancer Research Center, Seattle, WA), 293A (S. Reed, The Scripps Research Institute, La Jolla, CA), HeLa (American Type Culture Collection), and U2OS cells (American Type Culture Collection) were maintained in DME with 10% FBS and penicillin/streptomycin.

Techniques: Expressing, Knock-Out, Clone Assay, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

doi: 10.1083/jcb.200802076

Figure Lengend Snippet: c-Myc and SREBP1 regulation in Fbw7-null cells. (A) Lysates from two independent sets of targeted Hct116 cells were immunoblotted for c-Myc or Grb2 (see for Grb2 panel). (B) Fbw7 knockout Hct116 cells were treated with cycloheximide as indicated, and lysates were immunoblotted for c-Myc. (C) Real-time PCR analysis of c-Myc mRNA in the indicated Hct116 cells. Results are expressed as relative to wild-type Hct116 cells. Error bars show standard deviation (three replicates). (D) Lysates from the indicated Hct116 cells were immunoprecipitated with anti-SREBP1 antibody and immunoblotted for either total SREBP1 (left) or pT426 SREBP1 (right).

Article Snippet: Hct116 (J. Simon, Fred Hutchinson Cancer Research Center, Seattle, WA), human foreskin fibroblasts (W. Carter, Fred Hutchinson Cancer Research Center, Seattle, WA), 293A (S. Reed, The Scripps Research Institute, La Jolla, CA), HeLa (American Type Culture Collection), and U2OS cells (American Type Culture Collection) were maintained in DME with 10% FBS and penicillin/streptomycin.

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

doi: 10.1083/jcb.200802076

Figure Lengend Snippet: Isoform-specific cyclin E regulation by Fbw7. (A) Lysates from two independent clones of each Hct116 genotype were analyzed for cyclin E abundance and kinase activity (Grb2, loading control). (B) Pulse-chase analysis of cyclin E in Hct116 knockout lines. Cells were labeled 1 h after release from aphidicolin arrest (see text). Quantitation of the phosphorimage is depicted in the graph, and the primary data for each isoform-specific cell line is shown. (C) HeLa cells and 293A cells were transfected with the indicated Fbw7 siRNAs (α, β, γ, and CR) or control siRNAs (GFP and C). Cyclin E abundance, kinase activity, and T62 phosphorylation were measured 72 h after transfection. (D) 293A cells and HeLa cells were cotransfected with a FLAG-Fbw7γ expression vector in combination with the indicated siRNAs (γ-tubulin, loading control). (E) 293A cells were transfected with vectors expressing myc-tagged wild-type or P382I cyclin E. Lysates were immunoprecipitated with anti-Myc tag antibody (9E10), and cyclin E abundance, kinase activity, and T62 phosphorylation are shown.

Article Snippet: Hct116 (J. Simon, Fred Hutchinson Cancer Research Center, Seattle, WA), human foreskin fibroblasts (W. Carter, Fred Hutchinson Cancer Research Center, Seattle, WA), 293A (S. Reed, The Scripps Research Institute, La Jolla, CA), HeLa (American Type Culture Collection), and U2OS cells (American Type Culture Collection) were maintained in DME with 10% FBS and penicillin/streptomycin.

Techniques: Clone Assay, Activity Assay, Control, Pulse Chase, Knock-Out, Labeling, Quantitation Assay, Transfection, Phospho-proteomics, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

doi: 10.1083/jcb.200802076

Figure Lengend Snippet: Cyclin E sensitivity to Fbw7 varies during the cell cycle. (A) The indicated Hct116 cells were treated with either aphidicolin (S), nocodazole (G2M), or DMSO (A) for 16 h, and lysates were analyzed by Western blot (γ-tubulin, loading control). Cyclin E abundance is shown. In each case, >90% of the nocodazole-arrested cells were in G2M and >70% of the aphidicolin-arrested cells were in S phase (not depicted). (B) The indicated Hct116 cell clones were arrested in S phase with a sequential thymidine/hydroxyurea block and released for the indicated time periods. The abundance of cyclin E, phosphorylated cyclin E species, and Grb2 (loading control) are shown. (C) Hct116 cells were treated as in A and lysates were examined for Fbw7 and cyclin E expression. Fbw7-null cell lysates serve as negative control. (D) Hct116 cells were arrested and released from aphidicolin and Fbw7 abundance was determined.

Article Snippet: Hct116 (J. Simon, Fred Hutchinson Cancer Research Center, Seattle, WA), human foreskin fibroblasts (W. Carter, Fred Hutchinson Cancer Research Center, Seattle, WA), 293A (S. Reed, The Scripps Research Institute, La Jolla, CA), HeLa (American Type Culture Collection), and U2OS cells (American Type Culture Collection) were maintained in DME with 10% FBS and penicillin/streptomycin.

Techniques: Western Blot, Control, Clone Assay, Blocking Assay, Expressing, Negative Control

Journal: The Journal of Cell Biology

Article Title: Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

doi: 10.1083/jcb.200802076

Figure Lengend Snippet: Cyclin E–CDK2 activity varies during the cell cycle . (A) HeLa cells were transfected with either control or Fbw7-siRNA and then 48 h later were treated with nocodazole for 16 h. Mitotic cells were shaken off and replated for the indicated times. Cyclin E abundance and activity, as well as cyclin A abundance (a marker of cell cycle synchronization) are shown (*, background band). (B) Hct116 cells or Fbw7-null Hct116 cells were transfected with either control or p21Cip1-specific siRNAs and, after 48 h, treated with aphidicolin (Aph) or nocodazole (Noc). Cyclin E abundance, activity, and S384 phosphorylation are shown. The efficacy of p21 knockdown is shown. (C) Fbw7-null Hct116 cells were treated with aphidicolin or nocodazole and lysates were immunoprecipitated with anti–cyclin E antibody. The amount of CDK2 and Y15–phosphorylated CDK2 bound to cyclin E is shown. (D) Increased Fbw7 binding of cyclin E obtained from mitotic Fbw7-null lysates. Compare the amount of cyclin E that coprecipitates with Fbw7 in lanes 4 and 5 (middle).

Article Snippet: Hct116 (J. Simon, Fred Hutchinson Cancer Research Center, Seattle, WA), human foreskin fibroblasts (W. Carter, Fred Hutchinson Cancer Research Center, Seattle, WA), 293A (S. Reed, The Scripps Research Institute, La Jolla, CA), HeLa (American Type Culture Collection), and U2OS cells (American Type Culture Collection) were maintained in DME with 10% FBS and penicillin/streptomycin.

Techniques: Activity Assay, Transfection, Control, Marker, Phospho-proteomics, Knockdown, Immunoprecipitation, Binding Assay