PML plasmids encoding the RING domain (residues 49–104), B-box1 domain (residues 120–168), and RING + B-box1 (RB1, residues 49–168) as well as a UBC9 plasmid encoding the full-length protein (residues 1–158) were used to express these proteins in E. coli. All PML-coding genes were codon-optimized and chemically synthesized (GenScript). Coding regions of PML were cloned into the pGEX-6p-3 vector (GE Life Sciences). The encoded proteins thus include an N-terminal GST tag followed by an HRV 3C protease cleavage site. The plasmid encoding UBC9 was a gift from Cheryl Arrowsmith (Addgene plasmid # 25213; http://n2t.net/addgene:25213 ; RRID:Addgene_25,213). The encoded protein includes an N-terminal His6 tag followed by a thrombin cleavage site. The coding region of human SUMO1 was cloned into the pGEX-4 t-1 vector. The encoded protein includes an N-terminal GST tag followed by a thrombin cleavage site.
Pgex 6p 3 vector
Manufactured by GE Healthcare
Sourced in United Kingdom
The PGEX-6P-3 vector is a plasmid used for the expression of recombinant proteins in Escherichia coli. It contains a glutathione S-transferase (GST) tag sequence, which allows for the purification of the expressed protein using glutathione-affinity chromatography. The vector also includes a tac promoter, a multiple cloning site, and a pBR322 origin of replication.
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Lab products found in correlation
Glutathione sepharose 4b column, by GE Healthcare (3 mentions)
Superdex 200 pg, by GE Healthcare (3 mentions)
Hitrap q hp, by GE Healthcare (3 mentions)
Hitrap, by Cytiva (3 mentions)
Mouse monoclonal antibody isotyping test kit, by Bio-Rad (2 mentions)
Glutathione sepharose, by GE Healthcare (2 mentions)
Pcmv 3tag 6 vector, by Agilent Technologies (2 mentions)
Dpni, by New England Biolabs (2 mentions)
Bl21 star de3 cells, by Thermo Fisher Scientific (2 mentions)
Akta explorer 10, by GE Healthcare (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Peg4000, by Merck Group (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Bl21 star de3, by Thermo Fisher Scientific (1 mentions)
Pgex 5x 3 vector, by GE Healthcare (1 mentions)
Pegfp c1 vector, by Takara Bio (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Pcdna3 ha, by Thermo Fisher Scientific (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
18 protocols using pgex 6p 3 vector
1
Expression and Purification of PML and UBC9
2
Generating Anti-Monkey Podoplanin Antibodies
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A monkey podoplanin cDNA region encoding amino acids 76 to 89 (226–267 bp) was tandemly connected 21 times in a pGEX-6P-3 vector (GE Healthcare), replicated in BL21 (DE3) E. coli as the GST-tagged monkey podoplanin peptide (76–89 aa), and purified using glutathione sepharose (GE Healthcare). Six-week-old female BALB/c mice were immunized by neck and ventral subcutaneous injections of the GST-tagged peptide with Titer MAX Gold adjuvant (Titer MAX, Norcross, GA, USA). Cell fusion was performed by the procedure described previously [14 (link)]. Positive clones were subcloned three times by limiting dilution, and some were purified from ascites as described previously [48 (link)]. For the acute toxicity test with cynomolgus monkeys, large-scale purification of control IgG1 and 2F7 antibody was conducted. Six-week-old female BALB/c-nu/nu mice were injected intraperitoneally with 2F7-secreting hybridomas or control IgG1-secreting hybridomas (DIG104.10H.1) suspended in Hanks’ Balanced Salt Solution (HBSS, Gibco). Ascitic fluid was collected for purification of the antibody by salting out with ammonium sulfate and performing affinity column chromatography with protein G using AKTA Explorer 10S (GE Healthcare). The IgG isotype was determined with a Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Oxford, UK).
3
Generating Expression Constructs
4
Recombinant Expression of Rice PDIL Proteins
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Expression plasmids containing DNA fragments encoding the predicted mature-sized rice PDIL1;1 (GenBank: AK068268 ; Glu26−Leu512) and PDIL2;3 (GenBank: AK062254 ; Ser19−Leu441) were prepared as described previously [12] (link). The DNA fragment encoding the predicted mature-sized PDIL1;4 (GenBank: AK071514 ; Ser23−Leu563) was amplified from the full-length cDNA by PCR with a set of primers (5′-ataggatcctccgacgacgacctcga-3′ and 5′-tatctcgagtctagagagcgtcgttgc-3′) and inserted into pGEX-6P-3 vector (GE Healthcare) at the appropriate sites. Cys-to-Ala substitutions were introduced into the PDIL1;1 construct by means of a QuikChange II site-directed mutagenesis kit (Agilent Technologies) and the following sets of primers: Cys72Ala, 5′-ccgtggtgtggacacgccaagaagctcgctcc-3′ and 5′-ggagcgagcttcttggcgtgtccacaccacgg-3′; Cys414Ala, 5′-ccatggtgcggacacgccaagaagctggctcc-3′ and 5′-ggagccagcttcttggcgtgtccgcaccatgg-3′. The desired mutations were confirmed by DNA sequencing.
5
Monoclonal Anti-Podoplanin Antibody Generation
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A human podoplanin cDNA region encoding amino acids 76–89 (226–267 bp) was cloned and tandemly connected 12 and 40 times. These cDNA fragments were inserted into a pGEX-6P-3 vector (GE Healthcare, Buckinghamshire, UK). Next, the GST-tagged human podoplanin peptide (76–89 aa) produced in BL21 (DE3) E. coli was purified using glutathione sepharose. Six-week-old female BALB/c mice were injected with the GST-tagged peptide as an immunogen in conjugation with Titer MAX Gold adjuvant (Titer MAX, Norcross, GA, USA). Further, intraperitoneal immunization was performed intermittently for two months. Mice were euthanized, and splenocytes were fused with mouse myeloma P3U1 cells using PEG4000 (Merck, Whitehouse Station, NJ, USA). Hybridoma screening and antibody purification from ascites were performed as described previously [22 (link)]. IgG isotypes were identified using the Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Oxford, UK).
6
Pulldown Assay for Ten-m3 Interactors
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The intracellular domain of Ten-m3 was sub cloned into a pGEX-6P3 vector (GE Healthcare Life Sciences, Australia) which includes a Glutathione-S-transferase (GST) expression moiety. DNA was sequenced to confirm the orientation and correct reading frame for protein translation. Molecular weight of the fusion protein was estimated using Bioedit based on the amino acid composition and determined to be approximately 58 kDa (Ten-m3 insert ≈ 31.82 kDa; GST ≈ 26 kDa). Growth conditions for protein expression were determined empirically.
Ten-m3-GST and GST alone attached to sepharose beads were incubated with P1 whole brain total cell lysate. Fusion proteins attached to sepharose beads were retrieved by centrifugation, washed and resuspended in 2X Laemmli buffer pH 6.8 (20% glycerol, 4% SDS, 120 mM Tris–HCl, 10% β-mercaptoethanol, 0.02% (w/v) bromophenol blue). A third beads only control sample was prepared by resuspending sepharose beads in PBS.
Ten-m3-GST and GST alone attached to sepharose beads were incubated with P1 whole brain total cell lysate. Fusion proteins attached to sepharose beads were retrieved by centrifugation, washed and resuspended in 2X Laemmli buffer pH 6.8 (20% glycerol, 4% SDS, 120 mM Tris–HCl, 10% β-mercaptoethanol, 0.02% (w/v) bromophenol blue). A third beads only control sample was prepared by resuspending sepharose beads in PBS.
7
Expression Constructs for Mouse Brd4 and Interactors
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Expression construct for full-length mouse Brd4 has been previously described18 (link) and was based on pAdRSV-Sp vector with an N-terminal FLAG tag. Deletion of the C-terminal half of BRD4 (amino acids 603–1400) in the BRD4ΔC construct was generated by standard PCR techniques. GST-ET and GST-FLAG-NIPBL, generated by standard PCR techniques, were based on pGEX-6P-3 vector (GE Healthcare, Buckinghamshire, UK) and containing amino acids 609–718 of BRD4 and 212 to 449 of NIPBL, respectively. The siRNA molecules used for the different knockdowns were all obtained from Sigma-Aldrich (St. Louis, MO, USA), and are as follows: Nipbl siRNA #1, GCGAUAUACCCGUCUUGUU (SASI_Mm02_00351489); Nipbl siRNA #2, GGAAGAUUGGUAGCUUGUA (SASI_Mm02_00351487); Brd4 siRNA, GAGAAGGACAAGAAGGAAA; Control siRNA, CGUACGCGGAAUACUUCGA; Brd4 esiRNA, MISSION esiRNA EMU051511; Brd2 esiRNA, MISSION esiRNA EMU067621; Control esiRNA, MISSION esiRNA EHUFLUC. Control siRNA and esiRNA correspond to the Luciferase gene.
8
Purification of α-Catenin and Vinculin
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α-Catenin and vinculin were expressed and purified as reported in our previous study26 (link). DNA fragments of mouse αE-catenin M1-M3 (residues 276–634) and M2-M3 (residues 385–634) were amplified by PCR and cloned into the pGEX6P-3 vector (GE Healthcare). A DNA fragment of mouse full-length vinculin (residues 1–1066) was cloned into the pET-6b (+) vector (Novagen). These plasmids were verified by DNA sequencing and transformed into Escherichia coli strain BL21Star (DE3) cells (Invitrogen). α-Catenin and vinculin molecules were expressed at 20 °C in Luria-Bertani medium supplemented with 0.1 mM isopropyl-β-d -thiogalactopyranoside. BL21Star cells expressing α-catenin and vinculin were suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and disrupted by sonication. The supernatant after ultracentrifugation was applied to a Glutathione Sepharose 4B column (GE Healthcare). Proteins eluted from the column were further purified by anion exchange (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 200 pg, GE Healthcare) chromatography. N-terminal His6 tags on vinculin molecules were then cleaved using human rhinovirus 3C protease. For AFM structural imaging, GST-tags at N-termini of the wild-type and mutated fragments were cleaved by PreScission Protease (GE Healthcare).
9
Protein Phosphorylation Site Mutants
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pEGFP-HMW-MAP2 and pEGFP-NES-Jun were previously described (Björkblom et al., 2005 (link); Tararuk et al., 2006 (link)). Phosphorylation site mutants of HMW-MAP2, EGFP-HMW-MAP2T1619A/T1622A/T1625A (abbreviated to GFP-MAP2-AAA) and EGFP-MAP2T1619D/T1622D/T1625D (abbreviated to GFP-MAP2-DDD), were prepared by insertional overlapping PCR using mutagenic primers as previously described (Hongisto et al., 2008 (link)). The phosphorylation site numbering is based on the rat HMW-MAP2 Uniprot entry P15146. For in utero electroporation, the CMV promoter in EGFP-HMW-MAP2WT and EGFP-HMW-MAP2T1619D/T1622D/T1625D was changed to a CAG promoter for optimal expression in brain. MAP2C was isolated by PCR from rat brain cDNA. It was inserted downstream of GST in the pGEX-6P3 vector (GE Healthcare) using the pGEMTe cloning vector (Promega). pCDNA3-MKK7-JNK1 was a gift from Roger J. Davis (HHMI, Worcester, MA, USA).
10
Generating Expression Constructs
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Flag-tagged FLIPL, FLIPS, FADD and procaspase 8 expression constructs were generated in the pCMV-3Tag-6 vector (Agilent Technologies). GST-tagged FLIPS and FADD expression constructs were generated in the pGEX-6P-3 vector (GE Healthcare). Mutagenesis was carried out using the KOD Extreme polymerase (Novagen), with the template plasmid digested using DpnI (New England Biolabs).
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