Sc 74528

The chromatin immunoprecipitation (ChIP) assay was performed using the EZ-ChIP Chromatin Immunoprecipitation Kit (EMD Millipore) under the manufacturer's instructions. Forty-eight hours after infected with Ad-ANG, the SK-MES-1s cells were treated with fresh culture medium containing formaldehyde (formaldehyde final concentration 1%) for 10 minutes at room temperature on a rocking platform (80 rpm). Then the cells were treated with glycine solution (glycine final concentration 0.125mol/L) for 5 minutes. After washed by PBS, the cells were collected and lysed. DNA sonication was performed using a VCX400 ultrasonic processor (Sonics & Materials, Newtown, Conn). The immunoprecipitating antibody of ANG was anti- angiogenin antibody (Sc-74528, Santa cruz). For a negative control, normal mouse IgG in the EZ-ChIP Kit was used. The sequences of primers used were as follows: Forward 5'-TGAGTGCAATTGTGGTGTTAGG-3'; Reverse 5'-CTAGAGGCAACCGAAGTTCC-3' (amplification position: -847 to -947 bp upstream of the transcription start site of HMGA2). For semi-qPCR amplifications were performed with 35 cycles in a total volume of 20 μL and run on a 2% agarose gel. For RT-PCR the difference between the negative control and ANG varying at least 3 cycles was considered significant.