Takara mutanbest kit

In vitro site-directed mutagenesis was performed by using the TaKaRa MutanBEST Kit (TaKaRa, Dalian, China) following the instructions of the manufacturer. The plasmid pET-21b (+)-est816 was used as the template. The primers used were listed in SI Table S1. The correctness of the mutants was confirmed by DNA sequencing.

We designed a wild-type JUP CDS plasmid with HIS-tag in a pcDNA3.1+ vector. The R577C-JUP missense mutation was engineered into the vector using the Takara MutanBEST Kit (Takara Bio, Otsu, Shiga, Japan). AC16 cells were transfected with HIS-JUP-pcDNA3.1+ (WT and p.R577C) by using Lipofectamine™ 2000 CD Transfection Reagent (Thermo Fisher Scientific), following the manufacturer's instructions.

Technical-grade pyrethroids were provided by Jiangsu Yangnong Chemical Group Co., Ltd., Jiangsu, China. All ρ-nitrophenyl esters were purchased from Sigma. Restriction endonuclease, TaKaRa MutanBEST Kit, T4 DNA ligase and PrimeSTAR^® HS DNA Polymerase were purchased from TaKaRa (Dalian, China) and used according to the recommendations of the manufacturer. E.Z.N.A. Plasmid Mini Kit, E.Z.N.A. Gel Extraction Kit and E.Z.N.A. Yeast DNA Kit were purchased from OMEGA (Norcross, USA). GeneMorph^® II Random Mutagenesis Kit was purchased from Stratagene (La Jolla, CA, USA). Zeocin™ and EasySelect™ Pichia Expression Kit were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals and reagents were of analytical grade and were purchased from commercial sources, unless otherwise stated.

Empty-pEGFP-N1 vector, pEGFP-K-Ras^WT, pEGFP-K-Ras^G12V/T35S plasmids were transfected in cells. The transfection with specific gene with HA-tag was used for screening out the target factor through western blot. pEGFP-K-Ras^G12V/T35S plasmids were obtained by site-directed mutagenesis. SiRNAs (Shanghai GenePharma, Shanghai, China) refers to using interference RNA to silence the goal RNA (Mouse double minute 2 homolog (MDM2) or EYA3). The pEGFP-H2A.X^Y142A construct was constructed using the TaKaRa MutanBEST Kit (#D401) (TaKaRa, Shiga, Japan) as recommend by the manufacturer.

The potential binding sites of miR‐7‐5p with PAK2 were predicted by TargetScan database (http://www.targetscan.org/vert_71/). The human wild‐type (WT) PAK2 was amplified and cloned into luciferase reporter vector psiCHECKTM‐2 (Promega, Madison, WI, USA) to generate WT PAK2 plasmid. Meanwhile, PAK2 gene mutant 3′‐UTR recombinant plasmid (MUT PAK2) was generated using the TaKaRa MutanBEST Kit (TaKaRa, Beijing, China), which generated a mutation of 7 bps from GUCUUCC to ACUCCUU in the predicted miR‐7‐5p target binding site. Next, A549 or H1299 cells were seeded in 24‐well plates and cotransfected with 150 ng of WT or MUT PAK2 plasmid together with 50 nm miR‐7‐5p mimic or miR‐NC using Lipofectamine 2000. At 48 h after cotransfection, the activities of Firefly and Renilla luciferase were measured and relative luciferase activity was calculated with the Dual‐Luciferase Reporter Assay Kit (Promega).

Site-directed mutagenesis was performed with TaKaRa MutanBest kit (TaKaRa, Beijing, China). Briefly, the ilvC sequence was amplified by PCR from the cDNA template and PCR products were inserted into pOT2 plasmid. pOT2-Ilvc plasmid was amplified with designed mutant primers by PCR, and blunted using Blunting Kination Enzyme Mix, then ligated using ligation solution I in the kit. Plasmids were transformed into Escherichia coli Migula (Enterobacterales: Enterobacteriaceas) DH5α (TransGen, Beijing, China) and verified by DNA sequencing. Five amino acid residues contacting both NADP(H) and Mg²⁺ are conserved among bacteria, fungi, and plants; thus, their 5 active-site residue mutageneses were performed as mentioned above, respectively [15 (link)].
The recombinant ILVC protein was expressed in E. coli [16 (link)]. Briefly, ilvC with a 6× His-tag sequence at the C-terminus was amplified by PCR from the cDNA template or pOT2-Ilvc, and PCR products were inserted into pET-28b (+) vector (Novagen, Beijing, China), then transformed into E. coli strain BL21 (DE3)-competent cells (TransGen, Beijing, China). ILVC protein expression was induced by the addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM and purified with Ni-NTA agarose (Qiagen, Chatsworth, CA, USA) using a nickel-ion affinity column (Qiagen). Protein purity was monitored by SDS-PAGE.

The full-length RID1 cDNA and fragments of RID1 were cloned into the pGADT7 vector (Clontech, Palo Alto, CA, USA) at the EcoRI and XhoI sites to create the pAD-RID1 bait vector. RID1 fragments with mutant amino acid residues were created using a TaKaRa MutanBEST Kit (TaKaRa, Dalian, China). The pBD-GFA1 prey vector was a generous gift from Dr Yang Wei-Cai (Chinese Academy of Sciences, Beijing, China). Yeast transformation was carried out using a kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s protocol. The yeast strain used was Saccharomyces cerevisiae Y2H gold (Clontech, Palo Alto, CA, USA). Transformed cells were plated on SD-Trp-Leu/X-α-Gal/AbA for screening of positive colonies, and incubated at 30 °C for 3–5 d. Host yeast cells were transformed with the Empty AD/BD vectors used as negative controls.