HUVEC (ATCC® CRL-1730™) in 15cm2 dishes were fixed in 1% formaldehyde for 10 minutes and the fixation quenched by adding glycine to 125 mM for 5 minutes. Cells were washed twice in 1× PBS and harvested by scraping cells from the plate. ChIP was performed as described previously (Lee et al., 2006 (link)), with the exception that cellular extracts were sonicated in milliTUBE AFA Fiber tubes (Covaris, Woburn, MA, #520130) for 10 minutes in a Covaris S220 (Covaris, Woburn, MA) using the following conditions: Temperature: 5–9°C, Peak Power: 140 W, Duty Cycle: 5%, Cycles per Burst: 200. All ChIPs were performed using 500 μg of total extract and 2 μg of antibody per sample. Antibodies used were anti-Yap (Novus Biologicals, Littleton, CO, #NB110-58358) and normal rabbit IgG (Cell Signaling, Danvers, MA, #2729). Thirty microliters of Protein G Dynabeads (Invitrogen, Grand Island, NY, #100.02D) were used per ChIP. IgG and no antibody controls were routinely performed in parallel. After elution, ChIP DNA was analyzed by standard qPCR methods using a Step One Plus Real-Time PCR System (Applied Biosystems, Grand Island, NY).
ChIP qPCR primers:
ChIP qPCR primers: