Yap d8h1x

ChIP assays were performed according to the manufacturer’s instructions (Active Motif, CA). Briefly, freshly isolated intestinal epithelial cells were fixed with 1% formaldehyde, washed with cold PBS and lysed in lysis buffer. After sonication, protein-DNA complexes were incubated with YAP (D8H1X, Cell Signaling) or TEAD4 (sc-101184, SCBT) antibodies-coupled protein G beads at 4°C overnight, the normal rabbit and mouse IgG were used as the negative control. After elution and reverse cross-link, DNA was purified for subsequent PCR analysis. The primers used for real-time PCR of the promoter regions were described in Table S4. For real-time qPCR, total RNA was isolated using Trizol reagent (Invitrogen) and RNeasy Mini Kit (QIAGEN). cDNA was prepared using Superscript II Reverse Transcriptase (Thermo Fisher), and the amount of transcripts were quantified using Sybr Mastermix (Kapa Bioscience), with the respective oligonucleotides (Table S4) in Applied Biosystems 7300 RT-PCR systems. The number of copies of each gene was normalized to the housekeeping gene GAPDH. All qPCR experiments were conducted in biological triplicates, error bars represent mean ± standard deviation, and Student’s t test was used to generate p values (* = p value ≤ 0.05; ** = p value ≤ 0.01).

Total protein was extracted with RIPA lysis buffer. Nuclear and cytoplasmic fractions were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fischer, 78833). Protease inhibitors (Thermo Fischer, 87786) and phosphatase inhibitors (Thermo Fischer, 78420) were added. Antibodies against pYAP (Ser127) (D9W2I) (Cell Signaling, 13008), YAP (D8H1X) (Cell Signaling, 14074) and TBP (D5C9H) (Cell Signaling, 44059) were used at a 1:1,000 dilution in 5% BSA, overnight. Antibodies against β-actin (clone C4, Santa Cruz sc-47778) were used at a 1:2,000 dilution in 5% BSA for 2 h at room temperature.