Pcdna4 his maxb yap s127a

The expression plasmids for HA-Amot-p130, HA-Amot-p130^S176A and HA-Amot-p130^S176E were a gift from Dr. Hiroshi Sasaki (Kumamoto University, Japan). The following plasmids were previously described: psCMV-Pals1-Flag; PATJ-Flag (Wells et al., 2006 (link); Yi et al., 2011 (link)); Flag or HA-tagged Merlin (Kissil et al., 2002 (link), 2003 (link)); pCMV-Flag-Amot-p80, pCMV-V5-YAP and pCMV-Amot-130 LPxY/PPxY mutants (Yi et al., 2011 (link), Yi et al., 2013 (link)). The pcDNA4/His-MaxB-YAP-S127A (Plasmid #18988) and pCMV-Flag-YAP-5SA (Plasmid #27371) were from Addgene. Human Amot-p130 shRNA vectors have been previously described (Yi et al., 2011 (link)). siRNA duplexes targeting human YAP (ID #s20366) as well as non-targeting control siRNAs (ID #4390843) were from Thermo Fischer Scientific (Carlsbad, CA). The second siRNA targeting human YAP1 (5 FlexiTube #SI02662954) was purchased from Qiagen. siRNA targeting human angiomotin (ON-TARGETplus Human AMOT siRNA- smartpool L-015417-01-0005) or control pool of siRNA (ON-TARGETplus Non-targeting pool-set of 4 LU-017595-01-0002).

pcDNA4/HisMaxB mammalian expression vector was purchased from Invitrogen (#V864-20(43-0078F), USA). pcDNA4/HisMaxB-YAP1 (#18978) and pcDNA4/HisMaxB-YAP-S127A (#18988) were obtained from Addgene (Cambridge, MA, UK). In brief, prepared plasmids were amplified using Plasmid Plus Midi Kit (Qiagen, Hilden, Germany) and then selected by ampicillin (Sigma, MO, USA). Purified plasmid DNA was transfected using 4D-nucleofector X Kit for mammalian fibroblasts and Amaxa 4D-nucleofector (Lonza, Walkersville, MD, USA), following as manufacturer’s instructions. Zeocin selection reagent (#R250-01) (100 μg/ml) was used to select transfected cells. The transfection results were confirmed by DNA sequencing analysis (Bioneer, Co., Daejeon, Korea). In brief, DNA was extracted from WT-YAP-transfected and YAPS127A-transfected hTERT-hNOFs by using DNA Mini Kit (Qiagen, Hilden, Germany), and thereby RT-PCR was performed as indicated in RT-PCR method above. The following primer sequences to detect the mutation of phosphorylation site (serine at 127) in YAP; 5’- GTCATGAACCCCAAGACG -3’ (forward) and 5’- GGCAGAGGTACATCATCAGG-3’ (reverse). After electrophoresis, the exact DNA fragment was cut out with a scalpel and then DNA sequencing analysis was identified from Gel DNA extraction (iNtRON, Korea). Genomic identities were confirmed using BLASTN (http://www.ncbi.nlm.nih.gov/BLAST).