Lats2

Immunoblot was performed per a general western-blot protocol (Abcam). Antibodies for Flag-tag (#14793, 1:1000), HA-tag (#2367, 1:1000), Myc-tag (#2278, 1:1000), YAP (#14074, 1:1000), pYAP(S127, 1:2000) (#4911), LATS1 (#3477, 1:1000), LATS2 (#5888, 1:1000), pLATS1/2 (HM) (#8654, 1:1000), NCOR2 (#62370, 1:1000) and ERα (human) (#8644, 1:2000) were from Cell Signaling Technology. The GAPDH (#sc-25778, 1:3000), YAP/TAZ (#sc-101199, 1:2000) and ERα (mouse) (#sc-542, 1:1000) were obtained from Santa Cruz Biotechnology. The anti-Flag (HRP) (#A5892, 1:3000) was from Sigma.
For immunoprecipitations, cells were rinsed with ice-cold DPBS and then lysed in mild lysis buffer (150 mM NaCl, 50 mM Tris-Cl pH7.5, 0.5% Triton X-100) with protease inhibitors (Roche, #11873580001) and phosphatase inhibitors (Thermo Scientific, #88667) on ice for 30 min and centrifuged at 12,000 rpm for 10 min. Primary antibodies and Protein A/G magnetic beads (Pierce, #88803) were added to the supernatants and incubated with rotation for 3 h at 4 °C. Immunoprecipitants were washed four times with lysis buffer and were denatured by SDS-PAGE sample buffer and boiled for 5 min. Sample were followed by immunoblot analysis with antibodies indicated in the figures.

The indicated antibodies against the following proteins/peptides were used for Western blot analyses: Flag (F3165; Sigma-Aldrich), HA (MMS-101P; Covance), Myc (sc-40; Santa Cruz Biotechnology), GFP (sc-9996; Santa Cruz Biotechnology), WWP1 (H00011059-M01; Novus Biologicals), WWP2 (A302-935A; Bethyl Laboratories), AMOTL2 (sc-82501; Santa Cruz Biotechnology), USP9X (Abnova, H00008239-M01), CRB3 (NBP1-81185; Novus Biologicals), PALS1 (Santa Cruz, sc-365411), LATS1 (A300-477A; Bethyl Laboratories), LATS2 (#5888; Cell Signaling Technology), p-LATS1 (#8654; Cell Signaling Technology), YAP p-S127 (#4911; Cell Signaling Technology), YAP (H00010413-M01; Novus Biologicals), TAZ (#4883S; Cell Signaling Technology), CTGF (sc-14939; Santa Cruz Biotechnology), CYR61 (sc-13100; Santa Cruz Biotechnology), ubiquitin (550944; BD Biosciences), γ-tubulin (sc-7396; Santa Cruz Biotechnology), occludin (71-1500; Invitrogen), lamin B (sc-6217; Santa Cruz Biotechnology), and GAPDH (ab125247; Abcam); rabbit anti-AMOTL2 p-S159 was a kind gift from Dr Wanjin Hong (National University of Singapore). Antibodies used for immunofluorescence analyses were: YAP (H00010413-M01; same as for Western blot; Novus Biologicals), GFP (ab13970; Abcam), and BrdU (555627; BD Biosciences). The antibodies used for immunoprecipitations were the same as those used for Western blot analyses.

Antibodies to MARK2 (#9118), phospho-MARK activation loop (#4836) YAP/TAZ (#8418), pYAP S127 (#4911), pYAP S397 (#13619), LATS1 (#9153), LATS2 (#13646), pLATS1/2 Thr1079/1041 (#8654), MOB1 (#3863), phosphor-MOB1 (Thr35, #8699), MST1 (#3682), phosho-MST1/2 (Thr183/180, #3681)) and Myc Tag (#2278) were from Cell Signaling Technology (Danvers, MA, USA). HA antibody (#sc-7392) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-USP21, anti-MARK1, anti-actin, HA-, and Myc-conjugated beads were from Sigma-Aldrich (St Louis, MO, USA).

DMEM, FBS, goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488, Fura-2 AM and all RT-qPCR reagents were obtained from Invitrogen (Carlsbad, CA). Neurotensin, insulin and phalloidin-TRITC were obtained from Sigma Chemical (St. Louis, MO). The geranylgeranyl transferase I (GGTase I) inhibitor GGTI 298, PI 3-Kinase inhibitor A66 and the MEK inhibitor PD0325901 were from R&D Systems (Minneapolis, MN). The dual PI3K/mTOR inhibitor NPV-BEZ235 was purchasedfrom Selleck Chemicals (Houston, TX). Primary antibodies used were as follows: YAP (H-9, sc-271134 and 63.1, sc-101199, final dilution 1:200 for immunofluorescence), tubulin (sc-5274; final dilution 1:400), actin (sc-47778; final dilution 1:400) and GAPDH (sc-365062; final dilution 1:400) (Santa Cruz Biotechnology); phospho-YAP Ser127 (D9W2I, 13008; final dilution 1:1000), YAP (15028; final dilution 1:1000 for western blots), phospho-p70 S6 KinaseThr389) (9205; final dilution 1:1000) and phospho-S6 Ribosomal Protein Ser240/244 (5364; final dilution 1:1000), phospho LATS Thr1079 (8654; final dilution 1:1000) and LATS2 (5888; final dilution 1:1000) were from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase–conjugated anti-rabbit IgG and anti-mouse IgG were from GE Healthcare Bio-Sciences Corp (Piscataway, NJ). All other reagents were of the highest grade available.

Cells and tissues were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma-Aldrich). Protein lysates (20 μg) were run on SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% skim milk, and incubated overnight with primary antibodies at 4 °C followed by incubation with secondary antibodies (ZSGB-BIO). Proteins on membranes were visualized using the Chemiluminescent Reagents Kit (Millipore; Billerica, MA, USA). Signals were detected with the Chemi-Doc XRS+ (Bio-Rad; Hercules, CA, USA) and quantified using Image Lab 3.0 software (Bio-Rad). The following primary antibodies were used for western blotting: USP39 (#ab131332, Abcam), Histone H3(#ab176842, Abcam), Ki67 (#ab92742, Abcam), GAPDH (#sc-25778, Santa Cruz Biotechnology; Dallas, TX, USA), LATS1(#3477, Cell Signaling Technology), LATS2(#5888, Cell Signaling Technology), YAP (#14074, Cell Signaling Technology), and TAZ (#8418, Cell Signaling Technology).

Dasatinib, BIBW2992, PD0325091 and Etoposide were purchased from Selleck Chemicals (Houston, TX) and prepared as 20 mM stock solutions in DMSO. Antibodies used included anti-YAP-TAZ, pLats, Lats1, Lats2, pMST1/2, pH2AX, MEK, pMEK(S217/221), ERK, pERK(T202/Y204), pRb, pS6, LC3A/B, ATM, pChk1, Chk1, pChk2, Chk2, pCDC25c, pATRIP, p27, p21, pp38, β-catenin, cyclin D1, survivin, p4EBP1, pSMAD2(S465–467), p70S6K, pCDC2, CDC6, γH2AX, ATR, pDNA-PK(S2056) and DNA-PK, (Cell Signaling Technology, Danvers, MA); BRAF and cyclin E (Santa Cruz, Dallas, TX) and ATM, Flag M2 and β-actin (Sigma-Aldrich, St. Louis, MO); pATM(S1981) (GeneTex, Irvine, CA). Predesigned siRNAs of the target genes were purchased from Dharmacon (Pittsburgh, PA) or Thermo Scientific (Rodckford, IL). pcDNA4-Chk1-Flag and pCMV5-TOPO-3Xflag-TAZ plasmids were purchased from Addgene (Cambridge, MA). ^WTBRAF plasmid was provided by Dr. W. Kolch (Systems Biology Irland and The Conway Institute, University College Dublin).