Pcdna4 hismaxb yap1

pcDNA4/HisMaxB‐YAP1‐S127A (Addgene plasmid #18988), pcDNA4/HisMaxB‐YAP1 (Addgene plasmid #18978), and pCMV‐flag YAP2‐5SA (Addgene plasmid #27371) were obtained from Addgene. The pGL3 Basic luciferase reporter vector was purchased from Promega.

The biochemical reagents myricetin (#SM8390) and cisplatin (#D8810) were purchased from Solarbio (Beijing, China). MG132 (#M8699) was purchased from Sigma (St. Louis, MO, USA). The plasmids pcDNA4/HisMaxB-YAP1 (#18978), pCIneoMyc-LATS1 (#66851), and pCIneoMyc-LATS2 (#66852) were purchased from Addgene (Watertown, MA, USA). The following antibodies were used: anti-Caspase 3 (66470-1-Ig), YAP (13584-1-AP), c-Myc (#10828-1-AP), survivin (#10508-1-AP), CYR61 (#26689-1-AP), LATS1 (#17049-1-AP), LATS2 (#20276-1-AP), CTGF (#23936-1-AP) and Actin (#60008-1-Ig) (Proteintech Group, Chicago, IL, USA); p-YAP-S127 (#13008) (Cell Signaling Technology, Beverly, MA, USA); and p-LATS1/2-S909/872 (#AF8163) (Affinity Biosciences, Cincinnati, OH, USA).

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).