Plasmid construction

LATS-FLuc-BS was made as previously described (9). To generate LATS-NBiT-BS [LgBiT-YAP15-S127 (wild type [WT])/YAP15-S127A (mutant ([Mut]) and 14-3-3-SmBiT] in pcDNA3.1/hygro vector, PCR was performed using pBiT1.1-N [thymidine kinase (TK)/LgBiT] vector (Promega, Madison, WI, USA) and full-length cDNA of human 14-3-3τ (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NM_006826","term_id":"1519473540","term_text":"NM_006826"}}NM_006826) as template. The BS was cloned into the BamHI/NotI sites of pcDNA3.1/hygro(+). In order to make LATS-NBiT-BS in TK vector, oligos coding for YAP15-S127 [WT] and YAP15-S127A [Mut] were annealed and directly cloned into the pBiT1.1-N [TK/LgBiT] vector (Promega), and full-length 14-3-3 was amplified by PCR and cloned into pBiT2.1-C [TK/SmBiT] vectors (Promega) with the EcoRI/Bglll sites.

To make His-tagged fusion proteins, LATS-NBiT-BS in TK vector was used as template, and amplified LgBiT-YAP15 S127 [WT]/YAP15-S127A [Mut] and 14-3-3-SmBiT were cloned into the NdeI/BamHI sites of pET16b. Full-length cDNAs of human ALK (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_004304.4","term_id":"319803021","term_text":"NM_004304.4"}}NM_004304.4) was subcloned into pcDNA3.1/hygro(+)-myc vector. For lentivirus production, ALK cDNA was cloned into pTRIPZ lentiviral vector. For echinoderm microtubule-associated protein like 4 (EML4)-ALK cloning, cDNA was synthesized by reverse transcription from H3122 cells. EML4-ALK cDNA was amplified by PCR and cloned into pTRIPZ lentiviral vector. The following primers were used for cloning:

BamHI-kozak-LgBiT-YAP15-sense, 5′-CCGCGGATCCGCCGCCACCATGGTCTTCACACTCGAAGAT-3′; Not1-LgBiT-YAP15- antisense, 3′-CAAAGGAAGCGGCCGCTTACAACTGCAGAGAAGCTGGAGA-5′; BamH1-kozak-14-3-3-SmBiT-sense, 5′-CCGCGGATCCGCCGCCACCATGGAGAAGACTGAGCTGATC-3′; Not1-14-3-3-SmBiT-antisense, 3′-CAAAGGAAGCGGCCGCTTACAGAATCTCCTCGAACAGCCG-5′.

EcoR1-YAP-S127-BglII-sense, 5′-AATTCACCACAGCATGTTCGAGCTCATTCCTCTCCAGCTTCTCTGCAGTTGTGAA-3′; ECoR1-YAP-S127-BglII-antisense, 3′-ATCTTCACAACTGCAGAGAAGCTGGAGAGGAATGAGCTCGAACATGCTGTGGTG-5′; EcoR1-YAP-A127-BglII-sense, 5′-AATTCACCACAGCATGTTCGAGCTCATGCGTCTCCAGCTTCTCTGCAGTTGTGAA-3′; ECoR1-YAP-A127-BglII-antisense, 3′-ATCTTCACAACTGCAGAGAAGCTGGAGACGCATGAGCTCGAACATGCTGTGGTG-5′; BglII-14-3-3-sense, 5′-GGAAGATCTAATGGAGAAGACTGAGCTGATC-3′; ECoR1-14-3-3-antisense, 3′-CGCCGGAATTCCCGTTTTCAGCCCCTTCTGCC GC-5′.

NdeI-LgBiT-YAP15-sense, 5′-GGAATTCATATGATGGTCTTCACACTCGAAGATTTCGT-3′; BamHI-LgBiT-YAP15-antisense, (3′-CGCGGGATCCTTACAACTGCAGAGAAGCTGGAGAGGAATG-5′); BamHI-LgBiT-YAP15A127-antisense, (3′-CGCGGGATCCTTACAACTGCAGAGAAGCTGGAGACGCATGAGC-5′); NdeI-14-3-3-SmBiT-sense, (5′-GGAATTCATATGATGGAGAAGACTGAGCTGATCCAGAA-3′); BamHI-14-3-3-SmBiT-antisense, (3′-CGCGGGATCCTTACAGAATCTCCTCGAACAGCCGGTAGCC-5′).

Age1-kozak-ALk-sense, 5′-ACCACCGGTGCCGCCACCATGGGAGCCATCGGGCTCCTG-3′; MLU1-ALK-FLAG-antisense, 3′-ACACGATACGCGTTCACTTGTCGTCATCGTCTTTGTAGTCGGGCCCAGGCTGGTTCATG-5′. Age1-kozak-FLAG-BgLII-EML4-ALK-sense, 5′-AACCACCGGTGCCGCCACCATGGACTACAAAGACGATGACGACAAGGGAAGATCTATGGACGGTTTCGCCGGCAGT-3′; MLU1-N1-EML4-ALK-antisense, 5′ CGACGCGTATAAGAATGCGGCCGCTCAGGGCCCAGGCTGGTTCATGCT-3′.