Δ559, T670I and the new variant Δ574–580 mutant KIT receptors were obtained as following a methodology reported in our previous work [15 (link)–17 (link), 19 (link)]. Briefly, an expression vector carrying wild-type (WT) human complementary DNA (cDNA) for KIT (kind gift of Professor Y. Yarden, Weizmann Institute, Rehovot, Israel) was used to generate all mutated forms of KIT via site-directed mutagenesis using the commercial QuickChange Site-Directed Mutagenesis kit (Promega, Madison, WI), following manufacturer’s instruction. We then constructed mutant Δ559, in which amino acid 559 is removed from the juxtamembrane region by deleting nucleotides 1696 – 1698 from the portion of the exon 11-derived WT cDNA. For the T670I mutant, the second base of the T670 triplet codon ACA (i.e., cytosine 2030), was mutated to a thymine. Finally, the Δ574–580 mutant was obtained by removing nucleotides 1741–1761 from the corresponding portion of the WT cDNA. All plasmid inserts were sequenced after mutagenesis to verify their identity.
Quick change site directed mutagenesis kit
Manufactured by Promega
The Quick Change site-directed mutagenesis kit is a laboratory tool used to introduce targeted mutations into DNA sequences. The kit provides a straightforward method for creating specific changes in plasmid DNA without the need for subcloning.
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4 protocols using quick change site directed mutagenesis kit
1
Generation of Mutant KIT Receptors
2
Mutagenesis of OPN Promoter ERRα Sites
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A 834-bp human OPN promoter fragment was cloned into the kpn I / xho I sites of the luciferase reporter plasmid pGL3-basic (Promega) and named as OPN-luc. The primer sequences were as follows: forward, 5'-CGGGGTACCCATGGATGAGGGAACAAGG-3'; reverse, 5'-CCGCTCGAGTACCTTGGTCGGCGTTTGG-3'. The putative ERRα binding sites in the OPN promoter were mutated using the Quick Change site-directed mutagenesis kit (Promega). The primer sequences were as follows: Mut1, forward 5'-GCCCAAGGTTGCACATATTTGCAGTGACACAGCGGA-3'; reverse 5'-TCCGCTGTGTCACTGCAAATATGTGCAACCTTGGGC-3'. Mut2, forward 5'-AAAGCTAAGCTTGAGTAGTAGACCAGTGAGGCAAGTTTTCTG-3'; reverse 5'-CAGAAAACTTGCCTCCATGGTCTACTACTCAAGCTTAGCTTT-3'. Cells were incubated in triplicate in 24-well plates and transfected with the promoter constructs (200 ng) in combination with the plasmid pRL-TK (20 ng) as an internal control. Luciferase activity was examined with the Dual-Luciferase Reporter Assay System (Promega).
3
Generating Lentiviral Plasmids for p300 and p65
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pCi-p300 and pCi-PCAF are described elsewhere (Boyes et al., 1998 (link)). pcDNA3/myc-p300 and pcDNA3/T7-p65 were described previously (Chen et al., 2001 (link)). pCi-p300 Y1503A, F1504A and pcDNA3/T7-p65 K310R were constructed by using the QuickChange site-directed mutagenesis kit (Promega). Lentiviral plasmids, pCSII-CMV-MCS, pCAG-HIVgp, pCMV-VSV-G-RSV-Rev are kindly provided by H Miyoshi, RIKEN BioResource Center, Tsukuba, Japan (Bai et al., 2003 (link)). p300 CDS was cloned into XhoI/NotI site of pCSII-CMV-MCS.
4
Validation of miR-144-3p Regulation of ERO1L
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A fragment of ERO1L 3'-untranslated region (3'-UTR) containing the putative binding site for miR-144-3p was amplified from normal human genome DNA and cloned into the Spe I and Hind III sites of pMIR-REPORT firefly luciferase vector (Promega), named as ERO1L-WT. The primer sequences were as follows: forward 5'-GGACTAGTCTCCCTAATATCCTTCAGTGA-3'; reverse 5'-CCAAGCTTTTGAATACAATTTCAGCT-3'. ERO1L mutant 3'-UTR recombinant plasmid was generated using the Quick Change site-directed mutagenesis kit (Promega) with the primers: 5'-GTATGTCACTTAAATCGATGACAATTGTTTTATTTTTC-3' and 5'-GAAAAATAAAACAATTGTCATCGATTTAAGTGACATAC-3', which generated a mutation of 6 bps from TACTGT to ATGACA in the predicted miR-144-3p target binding site, named as ERO1L-Mut. All constructs were confirmed by DNA sequencing.
For reporter assay, cells were incubated in triplicate in 24-well plates and transfected with 200 ng promoter constructs (ERO1L-WT or ERO1L-Mut) together with 50 ng pRL-TK plasmids and 50 nM miR-144-3p mimics or miR-NC. 48 h later, luciferase activity was examined with the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity.
For reporter assay, cells were incubated in triplicate in 24-well plates and transfected with 200 ng promoter constructs (ERO1L-WT or ERO1L-Mut) together with 50 ng pRL-TK plasmids and 50 nM miR-144-3p mimics or miR-NC. 48 h later, luciferase activity was examined with the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to renilla luciferase activity.
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