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HEK293 cell lines were obtained from ATCC. Cell Lines were grown in Minimum Essential Media (MEM) supplemented with 10% FBS and grown at 37C with 5% CO2. For G1 cell cycle arrest, cells were grown in MEM media without Serum for 24 hours. For G2 cell cycle arrest, cells were treated with 330 nM of Nocodazole (Sigma) for 24 hours. All transfections were preformed using the calcium phosphate method. Briefly, cells were grown to a density of 1.2–2.0 × 106 cells per 6 cm plate. 5 ug of DNA was mixed to a volume of 450 uL and 50 uL of 2.5 M CaCl2 was added. 500 uL of BBS (0.05 M BES, 0.28 M NaCl, 0.0015 M Na2 HPO4, pH 7.0) was added and solution vortexed to mix. Cells were re-fed with fresh MEM supplemented with 2.5 uL/mL 25-hydroxycholesterol (Sigma). Precipitate was applied to cells and incubated for 4 hours prior to removal with PBS. Cells were re-fed with fresh MEM and incubated 24–48 hours. All plasmids used in this study are listed in Table 1.
HEK293 cells were transfected with pGC95 (control scrambled shRNA, 5'-CGTGCAAGGTCAGTACATGTTCAAGAGACATGTACTGACCTTGCACG) or pGC93 (5'-TGCTCAGGCTGCTGCATATTTCAAGAGAATATGCAGCAGCCTGAGCA). 24 hours after transfection, cells were re-fed with MEM supplemented with 5 ug/mL puromycin and selected for 48 hours. Post-selection, cells were grown for 7–10 days and single colonies were selected. Two representative clonal expansions (1A and 2F) of pGC93 are shown.
RNA was purified from cells using Qiagen RNeasy kits. After purification, RNA was normalized by concentration used as a template for stratascript reverse transcriptase following manufacturers protocols (stratagene). Reverse transcribed RNA was then used for PCR towards Csn5, β-Actin, cyclin F, Fbx4, Fbx7, Fbw7, and Skp2.
Native lysates were made by resuspending cell pellets in an equal volume of buffer A (25 mM TRIS (7.5), 150 mM NaCl, 0.3% Triton X-100, 1 mM EDTA and 1 mM DTT supplemented with 1 mM PMSF, 0.25 ug/mL pepstatin, and 5 ug/mL each of leupeptin, aprotinin, and chymotrypsin). Lysates were cleared by centrifugation and normalized using the Biorad Protein Assay (Biorad). Denatured lysates were made by resuspending cell pellets in an equal volume of boiling SDS buffer (2% SDS, 50 mM TRIS (7.5) and 5 mM DTT) and boiled for 5 minutes. Lysates were sonicated and cleared by centrifugation.
The following antibodies were used in this study: Fbw7 (Orbigen PAB-10563), cyclin F (Santa Cruz Biotechnology sc-952), cyclin E (Santa Cruz Biotechnology sc-198), Csn5 (Santa Cruz Biotechnology sc-9074), β-tubulin (Santa Cruz Biotechnology sc-9104) Fbx7 (Zymed 51–8000) and c-myc (Santa Cruz Biotechnology sc-42). Skp2 was generously provided by W. Krek and Fbx4 was generously provided by W. Boelens.
Native lysates were immunoprecipitated for 1 hour with antibodies toward cyclin E. Immunoprecipitates were washed 3 times in native lysis buffer (buffer A). Beads were resuspended in 10 uL of reaction mix (for 100 uL: 3 uL 5 mg/mL histone H1, 1.5 uL 4.5 mM ATP, 1.5 uL γ-32P ATP, 94 uL of kinase assay buffer (10 mM TRIS (7.5), 10 mM MgCl2, 50 mM NaCl, 2 mM EDTA, 1 mM DTT and 0.02% Triton X-100)). Reactions were incubated for 20 minutes at 21°C and analyzed by SDS-PAGE followed by autoradiography.
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