Pentr sd d topo vector

To prepare HvCPK2a constructs, cDNA clone 3093 NII, which encodes the barley kinase, was obtained from the National Institute of Agrobiological Sciences, Japan. HvCPK2a cDNA was amplified by Pfu DNA polymerase using the primers For 5′-CACCATGGGAAACTGCTGCGC-3′ and either N-Rev 5′-TAGCACGACGTCGCGCCGCTT-3′ or C-Rev 5′-CACGACGTCGCGCCGCTTCTT-3′, followed by cloning into the pENTR™/SD/D-TOPO® vector (Thermo Fisher Scientific) as either N- or C- terminal fusion constructs. To generate an N-terminal His-HvCPK2a fusion protein, the HvCPK2a clone was recombined with the Gateway® pDEST17™ vector (Invitrogen) using Gateway® LR Clonase® II Enzyme Mix (Invitrogen). To generate variants of HvCPK2a, a mutagenesis reaction was performed as described in Mitula et al. (2015 (link)) using the following primer pairs: K94M (For 5′-GCCTGCATGACCATCGCCAAG-3′, Rev 5′-CTTGGCGATGGTCATGCAGGC) and D189A (For 5′-CATCCACCGCGCCCTCAAGCC-3′, Rev 5′-GGCTTGAGGGCGCGGTGGATG-3′). The vector construct 35S:HvCPK2a-His-GFP (Earley et al., 2006 (link)) was used for Arabidopsis protoplast transformations and transient expression assays in Nicotiana benthamiana. To localize HvCPK2a and mutated versions of the protein in barley protoplasts, we generated a C-terminal HvCPK2a-GFP fusion in the pS5-DEST-EGFP vector using Gateway® LR Clonase® (Invitrogen).

The FBA promoter (2000 bp upstream of the starting ATG codon)
sequences were amplified from genomic DNA using iProof polymerase (Bio-Rad).
Promoters were cloned into the entry vector pDONR P4-P1r. The
β-glucuronidase (GUS) coding sequence was cloned into pENTR/SD/D-TOPO
vector (Thermo Fisher Scientific). The promoters were placed upstream of the GUS
coding sequence in the destination vector pB7m24GW using Multisite Gateway
cloning. The resultant promoter–GUS constructs were introduced into
wild-type Arabidopsis via Agrobacterium tumefaciens-mediated
transformation (Clough and Bent,
1998). Selection of transformants with BASTA was performed on soil-grown
seedlings.

Full length DNTTIP1, METTL21E and PARK7 cDNAs were amplified by reverse transcription PCR from total sheep RNA, directionally cloned into pENTR/SD/D-TOPO vector (Life Technologies, Grand Island, NY USA) and then subcloned by recombination into the pcDNA3.2/V5-DEST expression vector (Life Technologies), according to the manufacturer’s recommendations. The mouse pDLK1-pCMV-SPORT 6.1 plasmid was commercially available (cat#: MMM1013–9201636, Thermo Fisher Scientific Inc., PA USA). To confirm the insertion of the target genes with an intact open reading frame, all plasmids were sequenced from both directions. The pGWCAT-pcDNA3.2 /V5 control plasmid was obtained from Life Technologies.
The mouse Myh4 luciferase construct (pGL3IIB2.6) contains 2.56 kb of the promoter region of Myh4, and the rat Myh7 luciferase construct (p-3542β-MHCluc) contains 3.5 kb of the promoter of Myh7 [56 (link), 57 (link)]. The pRL-SV40 plasmid expressing renilla luciferase was commercially available from Promega Corporation. Plasmids for electroporation were purified using EndoFree Plasmid Maxi Kit (QIAGEN) and quantified by Nanodrop spectrophotometry (Thermo Fisher Scientific Inc., Rockford, IL USA.

The UUT1 coding region was synthesized into pUC57-Amp by Genewiz, amplified by PCR without the native stop codon, and introduced into the pENTR/SD/D-TOPO vector (Life Technologies) according to the manufacturer’s protocols to generate pENTR-UUT1. Recombination of the entry clone with destination vector pYES-DEST52 (Life Technologies) using LR clonase II (Life Technologies) produced a C-terminal His/V5 epitope fusion that was verified by sequencing before transformation into S. cerevisiae strain INVSc1 (Thermo Fisher Scientific). To verify heterologous protein expression, 2.5 µg of the proteoliposomes were resolved by SDS-PAGE and analyzed by immunoblotting with anti-V5 antibody (Thermo Fisher Scientific) as previously described (71 (link)). Reconstitution of microsomal proteins and transporter activity assays were also carried out as previously described (71 (link)). UDP-GlcA transport was measured at the UDP-GlcA concentrations and times indicated, and kinetic parameters were calculated by nonlinear regression using the Prism 6 application (GraphPad Software). Measured Uut1 content (see Table S2 in the supplemental material) was used to determine turnover rate.

The UXT1, UXT2, GMT1, and GMT2 coding regions were amplified from WT cDNA and introduced into the pENTR/SD/D-TOPO vector (Life Technologies) according to the manufacturer’s protocols to generate pENTR-UXT1, pENTR-UXT2, pENTR-GMT1, and pENTR-GMT2. Recombination of each entry clone with destination vector pYES-DEST52 (Life Technologies) using LR clonase II (Life Technologies) produced a C-terminal His/V5 epitope fusion that was verified by sequencing before transformation into S. cerevisiae strain INVSc1 (Thermo Fisher Scientific). Heterologous expression, reconstitution into proteoliposomes, and transport assays were performed as previously described [52 ]. UDP-Galf was prepared from UDP-galactopyranose (UDP-Galp) according to [53 (link)]. Protein expression and incorporation was verified by polyacrylamide gel electrophoreses and immunoblot analysis of 2.5 μg of microsomes or proteoliposomes using anti-V5 antibody (Thermo Fisher Scientific), also as previously described [52 ]. Kinetic parameters were calculated by non-linear regression using the Prism 6 application (GraphPad Sofware). The assay was validated and its sensitivity confirmed using the well-characterized GDP-Man transporters Gmt1 and Gmt2 (S9A Fig). Both proteins transported GDP-Man and smaller amounts of other GDP-sugars in exchange for GMP and, significantly less efficiently, UMP (S9D and S9E Fig).