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The rabbit antibodies against Amot were produced by Genemed Synthesis (San Antonio, USA) using the synthesized peptides (C-KTPIQILGQEPDAEMVEYLI) conjugated to keyhole limpet hemocyanin (KLH) for immunizations. Antibodies against GAPDH, Lamin A, cyclin D1, Rb, p-Rb as well as rabbit and mouse anti-Flag were from Santa Cruz BioTechnology; YAP, p-YAP, ERK, p-ERK, AKT, p-AKT from Cell Signaling Technology; PerCP mouse and APC mouse anti-BrdU from BD Transduction Laboratories.

For GFP-TEM4/GFP-LATS2 overexpression studies, cells were seeded at low density (50% confluency), in contrast to EAF1/PFKM overexpression studies were cells were seeded at high density (100% confluency). Both transfected and shRNA KD cells were lysed in 50 mM Tris-Cl, pH 7.6; 150 mM NaCl, 1% NP-40 supplemented with Protease inhibitors (cOmplete EDTA free and PhosStop phosphatase inhibitor, Sigma). Cells lysates were centrifuged for 10 min at 4oC to remove insoluble debris. Lysates were separated using 4%–20% Gradient gels (Miniprotein TGXTM Gel, Biorad) and immunoblotted using standard protocol. Primary antibodies used were YAP (4912S/Cell Signaling Technology, 1:1000), p-YAP (S127)(4911S/ Cell Signaling Technology, 1:1000) and GAPDH (MAB374/EMD Millipore, 1:5000). Blots were probed with mouse or rabbit specific IRDye 800 (LiCOR) and acquired, and quantified, using Odyssey Classic. Total RNA was isolated and purified from cells using miRNeasy Mini Kit (QIAGEN). cDNA was synthesized using the High Capacity RNA-to-cDNA kit (ABI) according to manufacturer’s instructions. qRT-PCR was performed using Fast SYBR Green Master Mix (ABI) on the QuantStudio 12K Flex Real-Time PCR System (ABI). Relative expression levels were defined using the DDCt method and normalizing to 36B4/RPLP0.

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.

Proteins were electrophoresed by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Next, the membranes were blocked in 5% skimmed milk and incubated with specific primary antibodies (1:1000) at 4 °C overnight. Rabbit anti-PTPRB (Biorbyt, Cambridge, UK), β-actin, E-cadherin, Vimentin, Slug, p-LATS1, p-YAP, LATS1, YAP, CTGF and CYR61 (Cell Signaling Technology) antibodies were used. After that, the membranes were incubated with the second antibody.

Total protein was extracted from GC tissues and cells by lysing in ice-cold lysis buffer. The proteins were electrophoresed on an SDS-PAGE gel, transferred to a PVDF membrane (Millipore, Danvers, MA, USA), and probed with a primary antibody targeted against CUL4A (Abcam, Cambridge, MA, USA), p21 (Abcam), p27 (Abcam), E-cadherin (Abcam), N-cadherin (Abcam), MST1 (Cell Signaling, Danvers, MA, USA), MST2 (Cell Signaling), LATS1 (Cell Signaling), YAP (Cell Signaling), p-YAP (Cell Signaling), vimentin (Cell Signaling), fibronectin (Cell Signaling) or β-actin (Cell Signaling). After incubating with the primary antibody, membranes were washed with TBS/0.05% Tween-20 (TBST) and incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) for 1 h at room temperature. After washing 3 times with (TBST) for 15 min, the membranes were developed using an ECL plus western blotting detection system.
For IP assays, HGC-27 cells were transfected with the Flag-LATS1 plasmid or co-transfected with Flag-LATS1 and HA-CUL4A plasmids, then treated with MG132 (10 μmoL) for 6 h, and lysed with the lysis buffer. Cell lysates were incubated with 5 μL of anti-HA beads (Catalog Number E6779, Sigma, USA) at 4°C for 4 h, then centrifuged and washed with RIPA buffer for 3 times. IP samples were immunoblotted in subsequent experiments.

Cell culture was performed as describe before^{37 (link)}. For cell proliferation assay, 5 × 104 cells were seeded in a 6-cm dishes in triplicate on day0 and counted on different time points indicated in the figures by cell counter (Nexcelom, USA). Cell proliferation curves were generated with GraphPad Prism software v.5.02 (La Jolla, CA, USA). For western blot, cells were lysed in standard RIPA buffer. Antibodies for NF2 (abcam ab88957), p-YAP (Cell Signaling 13008), YAP (Cell Signaling 12395), Slug (Cell Signaling 9585) and β-actin (Sigma A1978) were used for western blot.