Quikchange site directed mutagenesis kit

TargetScan software version 7.1 (www.targetscan.org) was used for bioinformatics analysis. To clarify the association between TINCR and miR-7, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent) was applied to generate the mutated miR-7-binding sites of TINCR. Then, the wild or mutated TINCR was sub-cloned into a Dual-luciferase Target Vector (Promega Corporation), generating the wild-type (WT) or mutated (MT) TINCR luciferase reporter plasmid, respectively. To clarify the association between miR-7 and mTOR, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent) was applied to generate the mutated miR-7-binding sites of mTOR 3′UTR. The WT or MT mTOR luciferase reporter plasmid was then generated. Cells were co-transfected as aforementioned with miR-7 mimic or negative control miR mimic (miR-NC), WT (or MT) TINCR reporter plasmid, WT (or MT) mTOR 3′UTR reporter plasmid. At 48 h after cell transfection, a Dual-Luciferase Reporter Assay System (Promega Corporation) was used to examine the luciferase activities. The ratio of firefly luciferase activity to Renilla luciferase activity was determined.

Human wildtype (ART-WT) and mutant (ARM) ARTEMIS expression plasmids were cloned via KpnI/NotI into pcDNA6/myc-His, pcDNA6V5-His or pcDNA4His/Max vectors (Invitrogen, Breda, Netherlands). The latter constructs in addition contained at the C-terminus a Myc Fusion tag, derived from pcDNA6/myc-His. Specific amino acid exchanges were introduced using either the QuikChange Site-Directed Mutagenesis Kit (Agilent) or the NEBuilder™ HiFi DNA assembly cloning kit (New England Biolabs). Forward and reverse primers used for amplification of deletion mutants and for mutagenesis are specified in Supplemental Tables S3 and S4, respectively. The nomenclature refers to the amino acids included in the respective ARTEMIS mutant proteins and specific amino acid exchanges, see (19 (link)) for details. Human DNA-PKcs fragments were cloned via KpnI/NotI into pcDNA4His/Max and in addition contained at the C-terminus a Myc Fusion tag, derived from pcDNA6/myc-His. The amino acid exchange L3062R was introduced via the QuikChange Site-Directed Mutagenesis Kit (Agilent). Forward and reverse primers used for amplification of deletion mutants and for mutagenesis are specified in Supplemental Tables S3 and S4, respectively. The nomenclature refers to the amino acids included in the respective DNA-PKcs fragment.

All plasmids were generated by standard molecular biology techniques. hhp1 and hhp2 genes including 500 base pairs upstream and downstream of the ORFs were amplified by PCR and ligated into a PCR-Blunt vector (Life Technologies) and then subcloned into a pIRT2 vector (Hindley et al., 1987 (link)). hhp1 and hhp2 mutants were created by mutagenizing pIRT2-plasmids containing hhp1⁺ and hhp2⁺ using a QuikChange site-directed mutagenesis kit (Agilent Technologies). Plasmids were validated by DNA sequencing.
The ORF of CK1δ was amplified by PCR from a plasmid (CK1δ pGEX-6p-2) kindly provided by Fanni Gergely (University of Cambridge). V405 4HA-CK1ε, a gift from David Virshup (Duke University Medical School) (Addgene plasmid #13724), was used as a template for PCR amplification of the CK1ε ORF. Each PCR product was cloned into pEGFP-C1, and the correct sequence was validated by DNA sequencing. Mutant CK1δ/ε variants were made using the QuikChange site-directed mutagenesis kit (Agilent Technologies) and confirmed by DNA sequencing.

Human TrkA^WT cDNA sequence (isoform II) in pReceiver-M03 (OmicsLink, ImaGenes, Berlin) was subcloned in pcDNA3.1 plasmid (Invitrogen). The mutation R649W was obtained starting from wild type sequence in pCDNA3.1, using the QuikChange site-directed mutagenesis kit (Agilent) and a pair of specific primers (Forward: CAT TTT GTG CAC TGG GAC CTG GCC ACA CGC; Reverse: GCG TGT GGC CAG GTC CCA GTG CAC AAA ATG). pCDNA3.1-human TrkA^WT and pCDNA3.1-human TrkA^R649W plasmids were transfected in Hek293 cells to perform western blot (WB) and ubiquitination assay described below.
The cloning to obtain S6-tagged human TrkA cDNA sequence in an ‘all-in-one’ third-generation Tet-on lentiviral pTRE vector has been described previously (49 ,102 ). This construct was used to generate S6-tagged human TrkA^R649W mutant, using QuikChange site-directed mutagenesis kit (Agilent) and the same pair of primers reported in the paragraph above. The mutant clone (S6-tagged TrkA^R649W) was checked by DNA sequencing and used for the transduction of immortalized and primary cells.

The TUG1 fragment containing the miR‐335‐5p binding site was amplified and cloned into the pmirGLO Vector (Promega, Madison, Wisconsin, USA) to gain the reporter vector pmiRGLO‐TUG1‐wild‐type (pmirGLO‐TUG1‐wt). The putative binding site of miR‐335‐5p in TUG1 was mutated by using a QuikChange Site‐Directed Mutagenesis Kit (Agilent, Santa Clara, California, USA) to synthetize pmirGLO‐TUG1‐mutant‐type (pmirGLO‐TUG1‐mut). The same method was used to obtain pmirGLO‐ROCK1‐wt and pmirGLO‐ROCK1‐mut reporter plasmids. The above plasmids were used for the following luciferase reporter assays. Similarly, the TUG1 fragment containing miR‐335‐5p binding site was amplified and cloned into the KpnI and XhoI restriction sites (Promega) of pcDNA3.1 vector to synthetize pcDNA3.1‐TUG1‐wild‐type (pcDNA3.1‐TUG1‐wt); pcDNA3.1‐TUG1‐mutant‐type (pcDNA3.1‐TUG1‐mut) was also gained by using a QuikChange Site‐Directed Mutagenesis Kit (Agilent). These two plasmids were used to construct TUG1 overexpression cell models.