The dimer formation of Stat5a was analyzed as described previously (4 (link)) and in the Supplementary Methods . Cells were serum-starved for 16 h, followed by pre-treatment with IST5-002 or the control compound (Ctrl) for 2 h at indicated concentrations, and stimulated with hPrl (10 nM) in serum-free medium for 30 min. Whole cell lysates were immunoprecipitated with anti-Myc mAb (2 μg/sample; Santa Cruz Biotechnology) and immunoblotted with anti-Flag mAb (1:1000; Genomics), anti-Myc mAb (1:1000; Santa Cruz Biotechnology) and anti-actin pAb (1:4000, Sigma-Aldrich).
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Stat5a Dimerization Assay Protocol
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Corresponding Organization : Sidney Kimmel Cancer Center
Other organizations : Georgetown University Medical Center, Georgetown University
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Variable analysis
independent variables
- IST5-002 concentration
- Control compound (Ctrl) concentration
- Stat5a dimer formation
- Serum starvation duration (16 h)
- HPrl concentration (10 nM)
- Stimulation duration (30 min)
- Anti-Myc mAb concentration (2 μg/sample)
- Anti-Flag mAb dilution (1:1000)
- Anti-Myc mAb dilution (1:1000)
- Anti-actin pAb dilution (1:4000)
- Positive control: Cells stimulated with hPrl (10 nM) for 30 min
- Negative control: Cells treated with control compound (Ctrl) for 2 h
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