Nxtract

Nuclear extracts were prepared from EL4 cells using NXTRACT (Sigma-Aldrich). In binding assays of 20 μl total volume, 28 μg of total nuclear extract from EL4 cells was incubated with 20 fmol of 3′ biotin-labeled oligonucleotide in the presence of 2 μl of 10× binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT [pH 7.5]) and 10 μM ZnSO4, 1 μl of 50% glycerol, 1 μg/μl poly(deoxyinosinic-deoxycytidylic) acid, 5 mmol/L MgCl₂ and 1% Nonidet P-40 at room temperature for 20 minutes. The oligonucleotides and methylated oligonucleotides (eurofins) were annealed and used as probes or competitors (Supplementary Table 2). All competitors were used at 200-fold excess. For supershift experiments, the nuclear extracts were incubated with 4 μg of anti-Kaiso Ab (6 F/6F8, ab12723; Abcam) and 5 μg of anti- HDAC3 Ab (ab7030; Abcam). The binding mixtures were loaded onto 6% native acrylamide gel (Thermo Fisher Scientific) in Tris/borate/EDTA buffer and electrophoresed for 50 minutes at room temperature under a constant 100 V. The gels were then transferred to a nylon membrane at 4 °C for 50 minutes under a constant 100V and exposed to UV light to crosslink for 15 minutes. The DNA binding activity was detected using a LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific). The image was obtained using an LAS-3000 IR.

Nuclear and cytoplasmic extracts were prepared using the SIGMA CelLytic NuCLEAR Extraction Kit as per manufacturer’s protocol. Briefly, cells were lysed in hypotonic lysis buffer and treated with 1% IGEPAL as per the manufacturer’s recommendations (NXTRACT, SIGMA) to obtain the cytoplasmic extract. The nuclear pellet was then washed thoroughly with lysis buffer and lysed in the extraction buffer. The protein concentration of each extract was determined by Bradford assay. Equal amounts of protein were resolved on a SDS-10% PAGE followed by western blot using the desired antibodies.

Nuclear and cytoplasmic proteins were extracted according to the manufacturer's instructions (Sigma, #NXTRACT). Western blotting was performed as described in detail in a previous study [26 (link)]. Primary antibodies used in current study included anti-mouse Yap1 (Abcam, #ab56701), TAZ (Cell signaling, #4883), Histone H4 (Santa Cruz, #sc-25260), Hsp90 (Santa Cruz, #sc-8262), p-Smad2 (Santa Cruz, #sc-101801), Smad3 (Cell signaling, #9513), p-Smad3 (Santa Cruz, #sc-11769), Krt14 (Santa Cruz, #sc-43310), Krt18 (Santa Cruz, #sc-45406) and p63 (Abcam, #ab124762).