Western blot, CHX, RT-qPCR, ChIP experiments were described previously (16 (link), 17 (link)). Antibodies for Western blots are Skp2 (Santa Cruz Biotechnology, H435), p21 (Santa Cruz Biotechnology, SC-397), p27 (BD Bioscience, #610242), p73 (Abcam, ab40658), activated Caspase 3 (Cell Signaling Technology, #9661), and α-tubulin (Sigma-Aldrich, T6074). RT-qPCR primers for p27 are sense: 5′-GCGGTGCCTTTAATTGGGTCT and antisense: 5′-GGCTTCTTGGGCGTCTGC T; p73 sense: 5′-AACGCCGAGCATCAATCC) and antisense: 5′-AGCCCAGACTCTGAGCACTT; GAPDH sense (5′-GGTTGTCTCCTGCGACTTCA and antisense 5′-GGTGGTCCAGGGTTTCTTAC. ChIP antibodies are E2F1 (Santa Cruz Biotechnology, SC-193X), E2F4 (Santa Cruz Biotechnology, SC-866X), IgG (Santa Cruz Biotechnology, SC-2027). ChIP primer sequences are: E2F1: sense 5-CTTTGGAGGTGAGCCTGAAGAG-3′, antisense 5′-GGGTCTGGCGAAGCGAACA-3′; p73: sense 5′-TGAGAGTGCGGTTCTATTGGC-3′, antisense 5′-GCCCTGAACATCTGCGTCTC-3′; GAPDH: sense 5′-GAGTTCTGGGAGTCTCGTGG-3′, antisense 5′-CTCTTCGGGTGGTGGTTCA-3′.
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Regulation of Cell Cycle and Apoptosis
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Other organizations : Albert Einstein College of Medicine, University of Pittsburgh
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Variable analysis
independent variables
- CHX (cycloheximide) treatment
- Protein levels of Skp2, p21, p27, p73, and activated Caspase 3 (measured by Western blot)
- MRNA expression levels of p27 and p73 (measured by RT-qPCR)
- Enrichment of E2F1, E2F4, and IgG (negative control) at E2F1 and p73 promoter regions (measured by ChIP)
- α-tubulin (used as a loading control for Western blot)
- GAPDH (used as a reference gene for RT-qPCR)
- Positive controls: None specified
- Negative controls: IgG for ChIP experiments
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