Yap sc 101199

Proteins from ileum samples and organoids were extracted using RIPA lysis buffer (P0013B, Beyotime, China) and separated on a 12% SDS-PAGE gel. The proteins were then transferred onto a nitrocellulose membrane, which was blocked with 5% bovine serum albumin for 1 h, and then incubated overnight with YAP (sc-101199, Santa Cruz Biotechnology) antibody, Phospho-YAP (Ser127) (13008 T, Cell Signaling Technology, USA) antibody, Nrf2 (16396-1-AP, Proteintech, China) antibody and GAPDH antibody (10494–1-AP, Proteintech, China) at 4 °C. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (SA00001-1, SA00001-2, Proteintech, China) for 1 h at room temperature. After the application of enhanced chemiluminescence (32209, Thermo Fisher Scientific, Inc., USA), imaging was done using the COMPLEX^TM2000 Automated Chemiluminescent Imaging System (Bioworld, Shanghai, China).

Antibodies to NEDD4 (Cat #2740), ITCH (#12117), SMURF1 (#2174), phosphor-YAP (S127, #4911), YAP (#4912), YAP/TAZ (#8418), phospho-LATS1 (T1079,#8654), LATS1 (#9153), LATS2 (#13646), specific K48 and K63 ubiquitin and Myc Tag (#2278) were from Cell Signaling Technology (Danvers, MA, USA). HA (#sc-7392) and YAP (sc#101199) antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-AMOT was from ABNOVA (Taipei, Taiwan). Anti-Flag antibody and anti-Actin, HA-, Myc- and Flag-conjugated beads were from Sigma-Aldrich, St Louis, MO, USA). DUB3 antibody (Cat#PA-5-44961) was obtained from Invitrogen (Taastrup, Denmark).

All cell lines were purchased from the ATCC and cultured under standard conditions. All cell-based assays were performed as described (Nguyen et al. 2017 (link); Nguyen et al. 2016 (link)). YAP localization was scored using mouse-anti-YAP (sc-101199, Santa Cruz) as previously described (Nguyen et al. 2017 (link)). Antibodies to phospho-YAP Ser127 (Cat #4911), YAP (#4912), TAZ (#2149), LATS2 #5888), phospho-LATS1/2 (#8654), Myc-Tag (#2272), LKB1 (#3050), MARK1 (#3319) and p-MARK1-4 (#4386) were from Cell Signaling. YAP (#sc-101199) antibody used for immunohistochemical staining was obtained from Santa Cruz Biotechnology. Anti-FLAG (M2, #F3165) and RhoBTB2 (SAB1407189) were from Sigma. Anti-actin (#MAB1501) was from Millipore.