To generate complementation or overexpression lines with otu5-1 or Col-0, 35S promoter and coding sequence of either wild-type OTU5a or OTU5a-C192S from entry vectors (pENL4-2-L3, pENTR221-OTU5a, and pENTR221-OTU5a-C192S, respectively) were mobilized together into gateway vector pKNGSTAP [85 (link)] using Gateway Technology Clonase II according to the manufacturer’s instruction (Invitrogen). The wild-type and C192S-mutated OTU5a coding sequences were first PCR-amplified using PfuTurbo (Agilent Technologies, Santa Clara, CA, USA) from pET28a-OTU5a and pET28a-OTU5a-C192S, respectively, with the primer pair OTU5a_CF and OTU5a_CR (Table S4 ) and cloned separately into pDONR221 (Life Technologies) to give pENTR221-OTU5a and pENTR221-OTU5a-C192S. The entry vector pENL4-2-L3 was requested from VIB, Belgium. The vector pET28a-OTU5a-C192S was derived by site-directed mutagenesis in accordance with the manufacturer’s instructions (Agilent Technologies) from pET28a-OTU5a, described previously [9 (link)] using the primer pair OTU5a-C192S-T and OTU5a-C192S-B (Table S4 ). A freeze–thaw method was used to transform Agrobacterium tumefaciens GV3101, and the A. thaliana transformation was performed as previously described [86 (link)]. Homozygous complementation and overexpression lines were selected from the T3 plants.
Pentr221
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PENTR221 is a laboratory instrument designed for the measurement and analysis of protein concentration. It utilizes a spectrophotometric method to determine the amount of protein in a sample. The core function of the PENTR221 is to provide accurate and reliable protein quantification data.
Automatically generated - may contain errors
Lab products found in correlation
Pdonr221, by Thermo Fisher Scientific (4 mentions)
Neb stable, by New England Biolabs (2 mentions)
Lr clonase 2, by Thermo Fisher Scientific (2 mentions)
Pmkate2 h2b, by Evrogen (2 mentions)
Bp clonase 2, by Thermo Fisher Scientific (2 mentions)
Pfuturbo, by Agilent Technologies (2 mentions)
Pad cmv v5 dest vector, by Thermo Fisher Scientific (1 mentions)
Adeno x rapid titer kit, by Takara Bio (1 mentions)
Adeno x maxi purification kit, by Takara Bio (1 mentions)
Pdest32, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Pef5 frt v5 dest, by Thermo Fisher Scientific (1 mentions)
10 protocols using pentr221
1
Generating Complementation and Overexpression Lines
2
Cloning and Tagging of Cyst Protein
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An ∼7.0-kb fragment (genomic region 19509164–19502181) encompassing the cyst gene was amplified from BACR27M12 (BACPAC) and recombined into pENTR221 (Invitrogen). To confer RNAi resistance on the resulting Entry Clone, silent mutations were introduced at two distinct sites corresponding to SH00146.N (TRiP). All other genomic constructs were derived from this Entry Clone. The GFP-tagged full-length construct was generated by inserting Drosophila codon-optimized superfolder GFP (Pédelacq et al., 2006 (link)) in between the ATG and the second codon of cyst. The GFP-tagged deletion constructs were generated by replacing the domains described in Fig. 6 A with superfolder GFP. In all constructs, an S(GGGGS)2 linker was introduced in between GFP and Cyst, and in CystΔN, the intron was left intact to preserve potential regulatory sequences. Finally, all Entry Clones were recombined into the Drosophila transformation vector pBID-G (Wang et al., 2012b (link)).
3
Engineered PB-TA-ERP2-mKate2-BRD4S Construct
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We engineered the PB-TA-ERP2-mKate2-BRD4S construct from two addgene clones (65378 [55 (link)] and 80477 [56 (link)]) and pmKate2-H2B (Evrogen, Russia). Overlap extension PCR was performed to amplify the mKate2 insert from the mKate2-H2B plasmid while adding the attB1 adapter and linker sequence. Another round of overlap extension PCR was performed to amplify the BRD4S insert from GFP-BRD4 while adding the attB2 adapter and linker sequence. A final round of fusion PCR was performed to fuse the fragments containing mKate2 and BRD4 to create an insert containing mKate2-linker-BRD4S. The primers used for the overlap extension PCR are listed in S3 Table . A BP reaction using BP Clonase II (Invitrogen, USA) was performed to clone the insert into pDONR221 (Invitrogen), creating the entry clone pENTR221-mKate2-BRD4S. The entry clone was amplified using NEB-Stable (New England Biolabs, USA). An LR reaction using LR Clonase II (Invitrogen) was performed with the destination vector PB-TA-ERP2 [56 (link)] to create the final expression vector.
4
Plasmid Vector for Mammalian Gene Expression
5
Adenoviral Overexpression of GADD45γ
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The complete open reading frame of human GADD45γ in pENTR221 (Invitrogen, Carlsbad, CA) was transferred to the pAd/CMV/V5-DEST vector (Invitrogen) by LR recombination. After sequencing to confirm the orientation and positions, the plasmids were transfected into HEK293A cells to produce recombinant adenoviruses. For controls, recombinant adenoviruses containing the lacZ gene were produced. For purification and concentration, the Adeno-X maxi purification kit (Clontech, Mountain View, CA) was used. For titration, HEK293 cells infected with recombinant adenoviruses were detected using an antibody specific for the adenovirus hexon protein with the Adeno-X rapid titer kit (Clontech). HRE cells were infected with adenoviral vectors harboring the GADD45γ gene or LacZ gene for 48 hours with a multiplicity of infection (MOI) of 25–50 before treatment with cisplatin or CsA.
6
Yeast Two-Hybrid Screening of AMPK Subunits
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Yeast two-hybrid screens were performed using the Gateway ProQuest Two-Hybrid System (Invitrogen) essentially as described by the manufacturer. To generate bait plasmids, AMPKα1 (encoding amino acids 10–559) and AMPKα2 (full length) in pENTR221 (Invitrogen) were transferred to the pDEST32 (Invitrogen) and transformed into MAV203 Saccharomyces cerevisiae strain. The ProQuest pre-made human fetal brain cDNA library and a human liver cDNA library in EXP-AD502 (Invitrogen) were transformed into pDEST32-AMPKα1 or pDEST32-AMPKα2 yeast strains, to perform screenings. AMPKα1/brain (4.2 × 106), AMPKα2/brain (2.9 × 106), AMPKα1/liver (2.1 × 106) and AMPKα2/liver (2.0 × 106) colonies were screened and positive clones were subjected to sequencing.
7
Engineered Ferroportin Variants for Functional Analysis
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The full‐length ORF clone pENTR221 (Invitrogen, Lofer, Austria) containing normal human ferroportin (IOH26826) was recombined with the expression vector pcDNA6.2/N‐EmGFP‐DEST (Invitrogen) using the Gateway LR Clonase Kit II (Invitrogen) as indicated by the manufacturer. Before recombining the IOH26826 vector with the expression vector pcDNA6.2/N‐EmGFP‐DEST, the following ferroportin mutations were introduced through site‐directed mutagenesis (SDM) into the ferroportin sequence: R178Q and A77D were used as ‘loss of function’ variants, whereas C326Y as a hepcidin‐resistant control.6 , 20 , 25 To create a human influenza hemagglutinin (HA) tagged ferroportin, the respective sequence was again inserted through SDM into the expression vector pcDNA6.2/N‐EmGFP‐DEST with a stop codon before the green fluorescent protein (GFP) sequence. SDM was exerted using the Q5 Site‐Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). The sequence of normal and mutant ferroportin constructs were confirmed by Sanger sequencing.
8
Engineered PB-TA-ERP2-mKate2-BRD4S Construct
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We engineered the PB-TA-ERP2-mKate2-BRD4S construct from two addgene clones (65378 54 and 80477 55 ) and pmKate2-H2B (Evrogen, Russia). Overlap extension PCR was performed to amplify the mKate2 insert from the mKate2-H2B plasmid while adding the attB1 adapter and linker sequence. Another round of overlap extension PCR was performed to amplify the BRD4S insert from GFP-BRD4 while adding the attB2 adapter and linker sequence. A final round of fusion PCR was performed to fuse the fragments containing mKate2 and BRD4 to create an insert containing mKate2linker-BRD4S. The primers used for the overlap extension PCR are listed in Supplementary Table 3. A BP reaction using BP Clonase II (Invitrogen, USA) was performed to clone the insert into pDONR221 (Invitrogen), creating the entry clone pENTR221-mKate2-BRD4S. The entry clone was amplified using NEB-Stable (New England Biolabs, USA). An LR reaction using LR Clonase II (Invitrogen) was performed with the destination vector PB-TA-ERP2 55 to create the final expression vector.
9
Alu/Alu Recombination Cassette Construction
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The Alu/Alu recombination puromycin (AARP) cassette was synthesized by Blue Heron Biotechnology, Inc (Bothell, WA) (http://www.blueheronbio.com ) and sub-cloned in pENTR 221(Life Technologies, Grand Island, NY). The pAARP/ENTR221 construct consists of two identical Alu elements (Ya5 consensus sequence). Alu1Ya5 is flanked by HindIII and BglII restriction sites, followed by a neomycin resistance gene flanked by NcoI and NdeI restriction sites, a SV40 poly A signal and an I-SceI restriction site. The second Alu element sequence, Alu2Ya5, is flanked by BamHI and EcoRI sites and followed by a puromycin resistance gene flanked by EcoRI and AgeI sites. The Alu/Alu recombination blasticidin (AARB) cassette was constructed by insertion of a blasticidin resistance gene (codon optimized bsr) at the EcoRI and AgeI sites (S1B Fig ). The Alu/Alu recombination eGFP (AARG) cassette was synthesized by Genescript Corporation (Piscataway, NJ), with eGFP sequence between the EcoRI and AgeI sites. pAARP/FRT_DEST, pAARB/FRT_DEST and pAARG/FRT_DEST were created in LR Clonase II plus reactions with 150 ng from each entry clone and 150 ng of pEF5/FRT/V5-DEST (Life Technologies). LR Clonase II plus reactions were performed as per the manufacturer’s protocol.
10
Synthesizing and Ligating Fno Plasmid
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A specific gene sequence unique to Fno [24 (link)] representing the FSC771 hypothetical protein gene (Genbank accession no. JQ780323.1) was synthesized and ligated into vector backbone pENTR221 (Geneart, Life Technologies Ltd, Paisely, United Kingdom). The resulting standard Fno-plasmid “pFNO STD”, (S1 Fig in supporting files) was transformed into an E. coli vector (OmniMAX™ 2 T1R) and purified from transformed bacteria using QIAprep8 Miniprep Kit (QIAGEN, UK). The final construct was verified by sequencing and the sequence congruence with in the insertion sites was 100%.
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