Phospho yap ser127

We selected typical sections of haematoxylin and eosin-stained slides that represent prominent intratumoral regions among the collected samples. From the invasive tumour front of each representative paraffin block, single core was obtained and transferred to a recipient TMA block.
Six types of immunostaining were performed on TMA using recommended doses of six different reagents according to the corresponding manufacturer’s instructions. Phospho-YAP (Ser127) (1:1000, monoclonal, D9W2I, #13008; Cell Signaling, Danvers, MA, USA), YAP (1:200, monoclonal, D8H1X, #14074; Cell Signaling, Danvers, MA, USA), KIBRA (1:200, polyclonal, ab216508; Abcam, Cambridge, MA, USA), LATS1/2 (1:200, polyclonal, #PA5-115498; Invitrogen, Carlsbad, CA, USA), Merlin (1:250, polyclonal, ab217016; Abcam, Cambridge, MA, USA), and MST1/2 (1:250, polyclonal, ab87322; Abcam, Cambridge, MA, USA) were used as primary antibodies. p-YAP, YAP, KIBRA, Merlin, and MST1/2 showed a diffuse staining pattern in the cytoplasm of cancer cells, whereas LATS1/2 showed a focal staining pattern in the cell nucleus. The staining level was read as 2-tiered. The stained tumour cells were graded as either positive or negative based on a higher intensity than that found in stromal cells and lymphocytes and an intensity equal to or lower than that of non-tumour cells, respectively.

Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium, EGF, LPA, verteporfin, crystal violet, poly-L-lysine, and anti-FLAG M2 Affinity agarose gel (A2220) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000, Lipofectamine RNAiMAX, Opti-MEM I, DAPI, goat serum, and Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). UNC3230, AR7, and 6AN were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies to α-tubulin (T5168), β-actin (A5316), vinculin (V4505), and FLAG-tag (F1804) were obtained from Sigma-Aldrich. Antibodies to HA-tag (#3724), Myc-tag (#2278), V5-tag (#13202), LATS1 (#9153), phospho-LATS1 Ser909 (#9157), YAP (#4912), phospho-YAP Ser127 (#4911), Merlin (#6995), and PIP5Kγ (#3296) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HA-tag (sc-7392), lamin B1 (sc-374015), Hsc70 (sc-7298), GFP (sc-9996), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

The following primary antibodies were used for immunoblot analysis: YAP, phospho-YAP (Ser127), Src, Lamine-b and PD-L1 from Cell Signaling, Inc. (Danvers, MA); phospho-YAP (Tyr357) from Abcam (Cambridge, MA) and Sigma-Aldrich (St. Louis, MO); TAZ and α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA). Total protein was extracted from cell lines using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Rockford, IL), and nuclear/cytoplasm proteins extracted using a nuclear/cytoplasm extraction kit (Thermo Fisher Scientific Inc.) were supplied with Complete Protease Inhibitor Cocktails (Roche, Lewes, UK), according to the manufacturers’ protocols. The protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). A total of 15 μg of proteins was run on 4∼20% gradient SDS–polyacrylamide gels (Bio-Rad) and transferred to Immobilon-P nitrocellulose membranes (Millipore, Bellerica, MA). The membranes were blocked in 5% non-fat milk and then probed with the primary antibodies overnight at 4°C. The membranes were incubated with appropriate secondary antibodies, and detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ).

Whole-cell extracts from cultured cells or tissues were prepared and subjected to western blotting. Equal amounts of cell extracts were resolved by 10% SDS-PAGE, analysed by immunoblotting and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Castle Hill, Australia). The following antibodies were used: anti-YAP (Cell Signaling Technology, 4912s), phospho-YAP (Ser127) (Cell Signaling Technology, #4911), β-actin (Santa Cruz, sc-1615), α-tubulin (Santa Cruz, sc-8035), PARP (Santa Cruz, sc-7150), Bcl-xl (Cell Signaling Technology, #2762s), CTGF(Abcam, ab6992), and Cyr61 (Gentex, GTX50042).

The following inhibitors or reagents were used in this study: LY294002 (Enzo Life Sciences, USA), MK-2206 (Invitrogen, USA), C3 exoenzyme (Cytoskeleton, USA), and pertussis toxin (PTX; Invitrogen, USA). YAP and phospho-YAP (Ser127) were purchased from Cell Signaling (Cell Signaling technology, USA). CTGF, ANG-2, and GAPDH antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA). Alexafluor secondary antibodies were from Life Technologies, USA.

Western blot analysis was carried out as previously described [30 ]. Primary antibodies used in western blot were listed as below: PLK4 (#NBP1-33042, Novus, USA), LATS1 (#3477, Cell Signaling Technology, USA), phospho-LATS1 (Thr1079) (#8654, Cell Signaling Technology), YAP (#14074, Cell Signaling Technology), phospho-YAP (Ser127) (#13308, Cell Signaling Technology), cleaved PARP (#5625, Cell Signaling Technology), γ-H2AX (Ser139) (#9718, Cell Signaling Technology), phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), phospho-ATR (Ser428) (#2853, Cell Signaling Technology), phospho-Chk1(Ser345) (#2348, Cell Signaling Technology), phospho-Chk2 (Thr68) (#2197, Cell Signaling Technology), anti-Histone H3 (#4499, Cell Signaling Technology). β-actin was used as a loading control. Chemiluminescent signals were detected using the Amersham Imager 600 imaging system (General Electric, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein bands normalized to control.