Kod plus neo dna polymerase

cDNAs for mouse CBX1, CBX3, CBX5, and CBX3 1/2 or 2/3 were generated by PCR using RNA from cultured MC of gddY mice with KOD Fx Neo or KOD plus neo DNA polymerases (Toyobo), and subcloned into a p3xFLAG-CMV-10 (Sigma-Aldrich) vector to produce proteins with N-terminal FLAG-tags. Human CBX3 cDNA was generated as described above using RNA from HEK293T cells, subcloned into p3xFLAG-CMV-10, or fused with linker peptide (G4S) and Strep-tagⅡ (WSHPQFEK) at the C terminus, and subcloned into a pcDNA3.4-TOPO vector (Thermo Fisher Scientific). To produce recombinant CBX3 in bacteria, CBX3 cDNA was subcloned into a pTrcHis2-TOPO vector (Sigma-Aldrich) containing a C-terminal 6× His-tag. To induce alanine mutations in CBX3 1/2, overlapping primers, each carrying a mutated codon, were used with either a primer at the 5′ or 3′ end of the cDNA fragment for the two-step PCR. The primers used are listed in Table 1.

The Escherichia coli strains used in this study were DH5α for plasmid propagation, and BL21 (DE3) star pRARE and Rossetta2 (DE3) pLysS (Invitrogen, Carlsbad, CA, USA) for protein expression. C. thermophilum was obtained from NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Tokyo, Japan). The protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Total RNA of C. thermophilum was obtained using the NucleoSpin RNA Plant (Takara Bio, Shiga, Japan), and cDNA was synthesized by the cDNA Synthesis Kit (Takara Bio). KOD-Plus-Neo DNA polymerase used for gene amplification and restriction endonucleases were obtained from TOYOBO (Osaka, Japan) and New England Biolabs Japan (Tokyo, Japan), respectively. Nucleotides and other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) or Sigma-Aldrich Japan (Tokyo, Japan). Actin was extracted from an acetone-dried powder made from chicken white breast meat, and was purified by ultracentrifugation, as previously described for rabbits [28 (link)]. Tubulin was purified from porcine brain with polymerization and depolymerization cycling [29 (link)]. CtCCT was expressed and purified as described previously [19 (link)].

Total RNA was isolated and first-strand cDNA was generated as described above. PCR was performed with the KOD-Plus-Neo DNA polymerase (Toyobo). Reaction products were analyzed on 1 to 2% agarose gels with ethidium bromide staining. The amount of each splicing isoform was quantified using ImageJ software. The primers used for splicing assays are shown in table S3.

The DNA fragment coding residues 1–119 of Bombyx mori ß-1,3-glucan recognition protein (GRP) and the cleavage site of HRV 3C protease (3C) was amplified with polymerase chain reaction using KOD plus neo DNA polymerase (TOYOBO CO., LTD. Osaka, Japan) with the primers 5′-CATGCCATGGAGTACGAGGCACCACCGGC-3′ and 5′-GGAATTCCATATGCGGGCCCTGAAACAGCACTTCCAGAAATTCTACTCCTGGTGTTATTTCAGAG-3′ from pET-GRP-3C-His as a template^{41 (link)}. The DNA fragment coding hFGF5 residues 21–242 [hFGF5 (21–242)] (GenBank id: NM_004464.3) was amplified with the primers 5′-CACCCATATGCACGGGGAGAAGCGTCTCG-3′ and 5′-GCCCTCGAGAGGGCTAGGTGGCTTTTTCTTTTCAG-3′ from Human Universal QUICK-Clone cDNA II (Takara Bio USA, Inc., CA, USA) as a template. DNA fragments of GRP-3C and hFGF5 (21–242) were cloned into pET-21d ( +) (Merck KGaA, Darmstadt, Germany) to give pET-GRP-3C-hFGF5 (21–242)-His.

FLAG-UBR5 (#37188), HA-GSK3β (#14753), HA-GSK3β-S9A (#14754), and HA-GSK3β-K85A expression plasmids were from Addgene (USA). FLAG-AMPKα1 (CH805185), FLAG-SKP2 (CH896343), and FLAG-AKT1 (CH846646) were purchase from Vigenebio (Shangdong, China). SIRT7 and GSK3β deletion mutants were constructed into the p3× FLAG-CMV10 vector (Sigma). GST-tagged SIRT7 or SIRT7-S259A/Thr263A/Thr255A was constructed by cloning into pGEX-4T-3 (GE Healthcare). The other SIRT7 mutants were generated by the site-directed mutagenesis using the KOD-Plus-Neo DNA polymerase (TOYOBO). All plasmids were confirmed by DNA sequencing (Genewiz). Details of the primers used for the generation of these plasmids or other experiments are shown in Supplementary Table 2.