Plasmids for expressing GST fusion proteins of Vps34 and p62 by inserting complementary DNA fragments of Vps34 and Mt (D285E), p62 and MT (S349A) into pGEX-2TK vector (GE Healthcare). GST-fused proteins were expressed in BL21 (DE3) E. coli cells by induction with 0.1 mM isopropyl thiogalactoside for 12 h at 30 °C. GST fusion proteins were affinity purified with glutathione-Sepharose resin (GE Healthcare) according to the manufacturer’s protocol.
Pgex 2tk vector
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
The PGEX-2TK vector is a plasmid that facilitates the expression of recombinant proteins in Escherichia coli. It contains a tac promoter for inducible protein expression, a glutathione S-transferase (GST) tag for affinity purification, and a thrombin cleavage site for tag removal.
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Lab products found in correlation
Glutathione sepharose resin, by GE Healthcare (1 mentions)
Glutathione high capacity magnetic agarose beads, by Merck Group (1 mentions)
Bl21 de3 bacterial strain, by New England Biolabs (1 mentions)
Histone h1, by New England Biolabs (1 mentions)
Nebuilder hifi dna assembly kit, by New England Biolabs (1 mentions)
M15prep4, by Qiagen (1 mentions)
Pqe40, by Qiagen (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
6 protocols using pgex 2tk vector
1
Expressing GST-Vps34 and GST-p62 Fusion Proteins
2
SLAMF1ct Protein Purification and Pulldown
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The GST fusion protein construct of SLAMF1ct (GST-SLAMF1ct) was prepared by cloning SLAMF1ct (corresponding with 259–335 aa of SLAMF1 protein; UniProtKB, Q13291 ) to pGEX-2TK vector (GE Healthcare) with the help of V. Kashuba (Karolinska Institute, Stockholm, Sweden). The sequenced plasmid was transformed into the BL21 DE3 bacterial strain (New England Biolabs, Inc.) or to the TKX1 strain (Agilent Technologies) for production of tyrosine-phosphorylated GST-SLAMF1ct-antiphosphotyrosine. Expression and purification of GST fusion proteins were performed as described previously (Shlapatska et al., 2001 (link)). For protein purification and pulldown assays, we used Glutathione high-capacity magnetic agarose beads (G0924; Sigma-Aldrich). Cell lysates of untreated and LPS-stimulated macrophages were prepared in 1× lysis buffer (0.5% NP-40, 150 mM NaCl, and 50 mM Tris-HCl, pH 8.0). Pulldowns were performed as described previously (Shlapatska et al., 2001 (link)).
3
Cloning and Characterization of Madcam1 Regulatory Sequences
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mCE-NFκB and mNFκB (−422 and −260 bp upstream of the Madcam1 transcription start site, respectively) were PCR-amplified as a KpnI-HindIII fragment from mouse genomic cDNA and cloned into pGL4.11[luc2P] to generate the luciferase (LUC) reporter constructs. To analyze the NKX2-3 and NR2F2 binding sites at CE, site-specific mutations were introduced into CE_ΔN-NFκB-LUC via dual PCR and for CE_ΔC_A, and CE_ΔC_AB via Cyagen custom service. CE-LUC, used for control in mutational studies, and human MADCAM1 reporter constructs hCE-LUC and hCE_NFκB-LUC were made by Cyagen custom service. Control Renilla (Ren) Luciferase vector is from Promega. For co-transfection studies, mammalian expression vectors Nkx2-3, Nr2f2 (COUP-TFII), and Hey1 were obtained from ABM custom service (Richmond, BC, Canada). All plasmids are described in Supplementary Table S3 .
Nr2f2, Hey1, and Nkx2-5 cDNA were PCR-amplified as BamHI-EcoRI-fragments and Nkx2-3 and Nkx2-3∆2-24 cDNA were PCR-amplified as EcoRI-HindIII- fragments from mouse cDNA and cloned into the pET21-A vector (Novagen) to generate His and T7-tagged recombinant proteins. Nr2f2 cDNA was cloned into the pGEX2TK vector (GE Healthcare) to generate GST-tagged COUP-TFII. pET21A-Nkx2-1 was from Cyagen, a custom service. The fidelity of all constructs was verified by sequencing. Primer sequences are provided in Supplementary TableS1 .
Nr2f2, Hey1, and Nkx2-5 cDNA were PCR-amplified as BamHI-EcoRI-fragments and Nkx2-3 and Nkx2-3∆2-24 cDNA were PCR-amplified as EcoRI-HindIII- fragments from mouse cDNA and cloned into the pET21-A vector (Novagen) to generate His and T7-tagged recombinant proteins. Nr2f2 cDNA was cloned into the pGEX2TK vector (GE Healthcare) to generate GST-tagged COUP-TFII. pET21A-Nkx2-1 was from Cyagen, a custom service. The fidelity of all constructs was verified by sequencing. Primer sequences are provided in Supplementary Table
4
Expression and Purification of GIP1 Protein
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Purified GIP1 was kindly donated by Nicolas Baumberger (IBMP, Strasbourg). GIP1 CDS was cloned into the pGEX‐2TK vector (GE Healthcare; courtesy of Etienne Herzog) and transformed into the BL21(DE3) E. coli strain. An overnight culture cultivated at 37°C was used to inoculate an expression culture at an OD600 of 0.1. The expression culture was grown at 37°C and 250 rpm until it reached an OD600 of 0.6. Afterwards, 0.5mM IPTG was added and the growth continued at 37°C for 6 h. Cells were collected by centrifugation at 5,000 g for 15 min and the pellet resuspended in 50 mM Tris pH 8, 300 mM NaCl, 5% glycerol, 5 mM EDTA, 0.1% Tween 20. Cells were lyzed by sonication and the lysate clarified by centrifugation at 10,000 g for 20 min at 4°C. GST‐GIP was purified by passage onto a glutathione‐sepharose GSTrap HP 1ml column (GE Healthcare) with 50 mM Tris pH 7.5, 10 mM MgCl2, 100 mM NaCl as an equilibration/washing buffer and 50 mM Tris pH 7.5, 10 mM MgCl2, 100 mM NaCl plus 10 mM reduced glutathione as an elution buffer. Elution fractions were analyzed on polyacrylamide gel and concentrated by ultrafiltration before being frozen and stored at −80°C. Histone H10 was purchased from NEB. In vitro kinase assays were performed as described previously (Harashima & Schnittger, 2012 (link)).
5
Generation and Validation of Obscurin and GTPase Constructs
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Human and chimeric obscurin GEF constructs 1–45 (cf. S2 , S3 and S9 Figs) have been generated with standard PCR techniques using the primers listed in S1 Table followed by transfer of PCR products into a modified pET15b vector (Novagen) with an N-terminal hexahistidine-tag and a TEV cleavage site using the NEBuilder® HiFi DNA Assembly kit (NEB, cat. no. E2621S). C-terminally truncated (for removal of prenylation site) small GTPases RhoA1-181,F25N, RhoB1-188,F25N, RhoC1-181,F25N, Rac11-177, Rac21-177, Rac31-177, RhoG1-177, Cdc421-178 and RhoQ/TC101-185 were cloned into a modified pGEX-2TK vector (GE Healthcare) in which the thrombin cleave-site was substituted by a TEV cleavage site. Cloning of constructs 46–58 (cf. S10 Fig ) and larg and vav2 DH domains was done by Bio Basic Inc (Canada, https://www.biobasic.com/ ). Cloning and viral packing of N-terminally eGFP-tagged human obscurin DH-PH (residues 5681–6019, numbering as in human obscurin B, NCBI ref. seq. NM_001098623) into either AAV9 or AdV vectors was performed by VectorBuilder Inc (United States, https://en.vectorbuilder.com/ ). All constructs were validated by sequencing.
6
Rabbit Antibody Production Against Mysm1
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This was performed according to previously described protocols.72 (link) Briefly, mouse Mysm1 cDNA fragment encoding amino acids 162–363 was cloned into pGEX-2TK vector (GE Healthcare, Buckinghamshire, UK), expressed as a GST fusion protein in Escherichia coli strain BL21 (New England Biolabs, Ipswich, MA, USA) and purified using affinity chromatography against glutathione sepharose 4B (GE Healthcare). Rabbit immunization was performed according to SOP 406 of the McGill Comparative Medicine and Animal Resource Centre (CMARC, http://www.mcgill.ca/research/researchers/compliance/ animal/sop), with 0.1 mg of protein injected subcutaneously in incomplete Freund's adjuvant, boosting every 4 weeks. For antibody purification, the same Mysm1 cDNA fragment was cloned into plasmid pQE40 (Qiagen), expressed as a DHFR fusion protein in E. coli strain M15(pREP4) (Qiagen), and was isolated using affinity chromatography against Ni-NTA agarose (Qiagen). Antibody purification from rabbit serum was performed by preparative immunoblotting, as previously described.72 (link)
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