Structured Review

GE Healthcare pgex 2t
Western-blot analysis of the SCP2 domain Left panel: proteins from various tissues within the last larval instar were used. Lane 1, midgut; lane 2, fat body; lane 3, Malpighian tubules; lane 4, recombinant protein corresponding to the SCP2 domain of SCPx. The antibody against the SCP2 domain of S. littoralis SCPx protein was used. Right panel: Western-blot analysis of the recombinant S. littoralis SCPx protein using anti-(SCP2 domain) antibody. Lane 1, proteins from E. coli carrying the <t>pGEX-2T</t> vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression.
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Images

1) Product Images from "Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans"

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

Journal: Biochemical Journal

doi: 10.1042/BJ20040717

Western-blot analysis of the SCP2 domain Left panel: proteins from various tissues within the last larval instar were used. Lane 1, midgut; lane 2, fat body; lane 3, Malpighian tubules; lane 4, recombinant protein corresponding to the SCP2 domain of SCPx. The antibody against the SCP2 domain of S. littoralis SCPx protein was used. Right panel: Western-blot analysis of the recombinant S. littoralis SCPx protein using anti-(SCP2 domain) antibody. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression.
Figure Legend Snippet: Western-blot analysis of the SCP2 domain Left panel: proteins from various tissues within the last larval instar were used. Lane 1, midgut; lane 2, fat body; lane 3, Malpighian tubules; lane 4, recombinant protein corresponding to the SCP2 domain of SCPx. The antibody against the SCP2 domain of S. littoralis SCPx protein was used. Right panel: Western-blot analysis of the recombinant S. littoralis SCPx protein using anti-(SCP2 domain) antibody. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Expressing

The developmental profile of SCPx during the sixth instar ( A ) A 10 #x3BC;g portion of total RNA from midgut at various time points within the sixth larval instar, as indicated, was used. The blot was hybridized with a 32 P-labelled SCPx cDNA probe corresponding to the open reading frame. The same blot was stripped and reprobed with 18S rRNA probe. G, W and PP refer to the stages ‘gut purge’, ‘wandering’and ‘prepupa’ respectively that occur before pupation. ( B ) Western-blot analysis of SCPx protein. Top panel: 100 #x3BC;g of protein from midgut at various time points within the sixth larval instar, as indicated, was used. Bottom panel: Western-blot analysis of the recombinant S. littoralis SCPx protein. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing the SCPx cDNA without induction of expression; lane 3, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression; lane 4, whole extract from C. elegans . The antibody against C. elegans P-44 was used to detect the SCPx protein.
Figure Legend Snippet: The developmental profile of SCPx during the sixth instar ( A ) A 10 #x3BC;g portion of total RNA from midgut at various time points within the sixth larval instar, as indicated, was used. The blot was hybridized with a 32 P-labelled SCPx cDNA probe corresponding to the open reading frame. The same blot was stripped and reprobed with 18S rRNA probe. G, W and PP refer to the stages ‘gut purge’, ‘wandering’and ‘prepupa’ respectively that occur before pupation. ( B ) Western-blot analysis of SCPx protein. Top panel: 100 #x3BC;g of protein from midgut at various time points within the sixth larval instar, as indicated, was used. Bottom panel: Western-blot analysis of the recombinant S. littoralis SCPx protein. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing the SCPx cDNA without induction of expression; lane 3, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression; lane 4, whole extract from C. elegans . The antibody against C. elegans P-44 was used to detect the SCPx protein.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Expressing

2) Product Images from "Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *"

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.561662

Validation of the modulative effect of candidate proteins in vitro . A , ORF of the four candidate E3 ubiquitin ligases chosen by the screen assay were cloned into the pGEX-2T bacterial expression vector, expressed as GST fusion proteins in E. coli , and
Figure Legend Snippet: Validation of the modulative effect of candidate proteins in vitro . A , ORF of the four candidate E3 ubiquitin ligases chosen by the screen assay were cloned into the pGEX-2T bacterial expression vector, expressed as GST fusion proteins in E. coli , and

Techniques Used: In Vitro, Clone Assay, Expressing, Plasmid Preparation

3) Product Images from "Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans"

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans

Journal: Biochemical Journal

doi: 10.1042/BJ20040717

Western-blot analysis of the SCP2 domain Left panel: proteins from various tissues within the last larval instar were used. Lane 1, midgut; lane 2, fat body; lane 3, Malpighian tubules; lane 4, recombinant protein corresponding to the SCP2 domain of SCPx. The antibody against the SCP2 domain of S. littoralis SCPx protein was used. Right panel: Western-blot analysis of the recombinant S. littoralis SCPx protein using anti-(SCP2 domain) antibody. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression.
Figure Legend Snippet: Western-blot analysis of the SCP2 domain Left panel: proteins from various tissues within the last larval instar were used. Lane 1, midgut; lane 2, fat body; lane 3, Malpighian tubules; lane 4, recombinant protein corresponding to the SCP2 domain of SCPx. The antibody against the SCP2 domain of S. littoralis SCPx protein was used. Right panel: Western-blot analysis of the recombinant S. littoralis SCPx protein using anti-(SCP2 domain) antibody. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Expressing

The developmental profile of SCPx during the sixth instar ( A ) A 10 #x3BC;g portion of total RNA from midgut at various time points within the sixth larval instar, as indicated, was used. The blot was hybridized with a 32 P-labelled SCPx cDNA probe corresponding to the open reading frame. The same blot was stripped and reprobed with 18S rRNA probe. G, W and PP refer to the stages ‘gut purge’, ‘wandering’and ‘prepupa’ respectively that occur before pupation. ( B ) Western-blot analysis of SCPx protein. Top panel: 100 #x3BC;g of protein from midgut at various time points within the sixth larval instar, as indicated, was used. Bottom panel: Western-blot analysis of the recombinant S. littoralis SCPx protein. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing the SCPx cDNA without induction of expression; lane 3, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression; lane 4, whole extract from C. elegans . The antibody against C. elegans P-44 was used to detect the SCPx protein.
Figure Legend Snippet: The developmental profile of SCPx during the sixth instar ( A ) A 10 #x3BC;g portion of total RNA from midgut at various time points within the sixth larval instar, as indicated, was used. The blot was hybridized with a 32 P-labelled SCPx cDNA probe corresponding to the open reading frame. The same blot was stripped and reprobed with 18S rRNA probe. G, W and PP refer to the stages ‘gut purge’, ‘wandering’and ‘prepupa’ respectively that occur before pupation. ( B ) Western-blot analysis of SCPx protein. Top panel: 100 #x3BC;g of protein from midgut at various time points within the sixth larval instar, as indicated, was used. Bottom panel: Western-blot analysis of the recombinant S. littoralis SCPx protein. Lane 1, proteins from E. coli carrying the pGEX-2T vector without the SCPx cDNA; lane 2, proteins from E. coli carrying the pGEX-2T vector containing the SCPx cDNA without induction of expression; lane 3, proteins from E. coli carrying the pGEX-2T vector containing SCPx cDNA with induction of expression; lane 4, whole extract from C. elegans . The antibody against C. elegans P-44 was used to detect the SCPx protein.

Techniques Used: Western Blot, Recombinant, Plasmid Preparation, Expressing

4) Product Images from "M-Like Proteins of Streptococcus dysgalactiae"

Article Title: M-Like Proteins of Streptococcus dysgalactiae

Journal: Infection and Immunity

doi:

Schematic presentation of deduced gene products encoded by the pDEMA6 clone derived from S. dysgalactiae 8215. pDEMA1 to pDEMA5 are phagemid clones isolated after panning a phage display library of strain 8215 against Fg. pGDEMA7 and pGDEMA8 are expression clones of the pGEX-2T vector with inserts corresponding to the phagemid clone pDEMA5 and a PCR fragment derived from pDEMA1 encoding amino acids 41 to 518 of DemA, respectively, yielding GST fusion proteins. The figures within parentheses after the names of the clones indicate the encoded amino acids of protein DemA. The DemA protein, including the signal peptide, consists of 548 amino acid residues as indicated above the schematic drawing. S, signal peptide, followed by the cell surface exposed part of the protein with repetitive sequences marked by different patterns; W, the cell wall spanning region; M, the membrane spanning region directly followed by five charged amino acids; ΔDmgA, a truncated protein highly homologous to the Mga regulatory protein from S. pyogenes ); ΔIS, 276 bp homologous to an IS element. +, the phagemid clone can bind Fg, human IgG, and bovine IgG; −, no detectable binding.
Figure Legend Snippet: Schematic presentation of deduced gene products encoded by the pDEMA6 clone derived from S. dysgalactiae 8215. pDEMA1 to pDEMA5 are phagemid clones isolated after panning a phage display library of strain 8215 against Fg. pGDEMA7 and pGDEMA8 are expression clones of the pGEX-2T vector with inserts corresponding to the phagemid clone pDEMA5 and a PCR fragment derived from pDEMA1 encoding amino acids 41 to 518 of DemA, respectively, yielding GST fusion proteins. The figures within parentheses after the names of the clones indicate the encoded amino acids of protein DemA. The DemA protein, including the signal peptide, consists of 548 amino acid residues as indicated above the schematic drawing. S, signal peptide, followed by the cell surface exposed part of the protein with repetitive sequences marked by different patterns; W, the cell wall spanning region; M, the membrane spanning region directly followed by five charged amino acids; ΔDmgA, a truncated protein highly homologous to the Mga regulatory protein from S. pyogenes ); ΔIS, 276 bp homologous to an IS element. +, the phagemid clone can bind Fg, human IgG, and bovine IgG; −, no detectable binding.

Techniques Used: Derivative Assay, Clone Assay, Isolation, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Binding Assay

Related Articles

Clone Assay:

Article Title: M-Like Proteins of Streptococcus dysgalactiae
Article Snippet: .. For additional cloning, the plasmid vectors pUC19 and pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) were used. .. Restriction endonucleases and Taq DNA polymerase were either from Amersham Pharmacia Biotech or MBI Fermentas (Vilnius, Lithuania).

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: .. ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The resulting PCR product was gel-purified and cloned into pGEM®-T Easy vector. .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: All other LSF substitution mutant cDNAs were cloned into the retroviral background by amplifying mutant LSF cDNA from the respective pCMV-LSF, digesting the amplicon with BamHI and XhoI and ligating it into BamHI- and XhoI-digested pLZRSpBMN-linker-IRES-EGFP. .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: .. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. .. A C-terminally extended construct of TOCA1 comprising residues 330–545 was cloned into pMAT10-P.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ). .. ADI, TF, SpyCEP, SLO, and SCPA antigens were expressed in E. coli BL21 Star (DE3) cells and purified by immobilized metal ion affinity chromatography (IMAC).

Article Title:
Article Snippet: .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare). .. The anti-DYRK1A antibody used in this study was raised by immunizing rabbits with a purified GST-fusion protein making up the last 144 amino acids of DYRK1A.

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Article Title: Biochemical and Genetic Analyses Provide Insight Into the Structural and Mechanistic Properties of Actin Filament Disassembly by the Aip1p-Cofilin Complex in Saccharomyces cerevisiae
Article Snippet: .. Vector pBH360 was created by cloning a cofilin PCR product into the Bam HI and Eco RI sites of pGEX-2T (GE Healthcare). ..

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: .. Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Centrifugation:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. Bacterium cells were harvested 5 h after induction, resuspended in buffer R (50 m m Tris-HCl (pH 7.5), 150 m m NaCl, and 1 m m EDTA), and lysed in buffer L (50 m m Tris-HCl (pH 7.5), 150 m m NaCl, 5 m m EDTA, 0.028% β-mercaptoethanol, and 0.4 mg/ml of lysozyme (Sigma)) at 4 °C for 1 h. After centrifugation, GST fusion proteins in the supernatant were bound to a glutathione-Sepharose 4B column (GE Healthcare), washed with buffer G (50 m m Tris-HCl (pH 8.0), 500 m m NaCl, 10% glycerol, and 5 m m DTT), and eluted with buffer G containing 15 m m reduced glutathione.

Amplification:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: .. ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Article Title: A novel splicing regulator shares a nuclear import pathway with SR proteins
Article Snippet: .. The transportin cDNA (from J.Steitz, Yale University, New Haven, CT) was amplified by PCR and inserted in-frame into pGEX-2T (Amersham Pharmacia Biotech). ..

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The cDNA containing the entire open reading frame was amplified by PCR with the following gene specific primers: ORF-F (5′-CGG ATC CCC TAG AAA AGT GTT CGT T) and ORF-R (5′-CGA ATT CTC ACA GTT TGG AGC GGA TTG T). .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: All other LSF substitution mutant cDNAs were cloned into the retroviral background by amplifying mutant LSF cDNA from the respective pCMV-LSF, digesting the amplicon with BamHI and XhoI and ligating it into BamHI- and XhoI-digested pLZRSpBMN-linker-IRES-EGFP. .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. .. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SpyCEP (amino acids 40 to 683, D151A S617A) sequence was cloned into the pET151/D-TOPO vector (Invitrogen) following amplification from GAS 5448 genomic DNA and QuikChange site-directed mutagenesis (Agilent Technologies). .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title:
Article Snippet: The human 14-3-3β open reading frame was amplified by polymerase chain reaction (PCR) from the pACTII-14-3-3β plasmid isolated from the cDNA library used in the yeast two-hybrid screen (human fetal brain library in pACTII; Clontech, Mountain View, CA). .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: During this procedure, the 4.4-kb region upstream from the sMMO operon was amplified by PCR from pTJS170 with the M13 forward sequencing primer (3′-GTA AAA CGA CGG CCA GT-5′) and the oligonucleotide 5′-G ACG CGT GGA TCC GAT CGT CGT ATG GCG ATG C-3′ ( Bam HI site is underlined). .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI.

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: .. Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Filtration:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. Eluted fractions containing GST fusion proteins were further subjected to gel filtration chromatography using a Superdex 200 column (GE Healthcare) with buffer G. Preparation of BAF (non-tagged) and GST-tagged VRK1 by E. coli were performed as described previously ( , ).

Construct:

Article Title: M-Like Proteins of Streptococcus dysgalactiae
Article Snippet: The phagemid vector pG8H6 ( ) was used to construct the phage display library. .. For additional cloning, the plasmid vectors pUC19 and pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) were used.

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: Paragraph title: Plasmid Constructs ... The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: Paragraph title: Expression Constructs ... Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively.

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: Paragraph title: Constructs, Recombinant Proteins, Antibodies, and Other Materials ... For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Enzyme-linked Immunosorbent Assay:

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ). .. To prepare antigens for ELISA, tobacco etch virus (TEV) protease was used to cleave the His tag from purified ADI, TF, SpyCEP, and SCPA; uncleaved protein and TEV were removed by IMAC.

Luciferase:

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: The Renilla luciferase expression plasmid, phRLTK was purchased from Promega. .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Expressing:

Article Title: M-Like Proteins of Streptococcus dysgalactiae
Article Snippet: E. coli DH5α [ supE44 ΔlacU169 (φ80 lacZΔM15 ) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ] was used for the expression of recombinant proteins. .. For additional cloning, the plasmid vectors pUC19 and pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) were used.

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Article Title: Successful expression and purification of DPPD using a codon optimized synthetic gene *
Article Snippet: These included various vectors such pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-2T (GE Healthcare, Piscataway, New Jersey). .. However, as mentioned before, low levels of rDPPD could be expressed and purified by using the Mycobacterium expression vector pSMT3 and Mycobacterium smegmatis as host cell [ ].

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: Paragraph title: Recombinant SCPx protein expression ... The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences. .. Bacterial expression plasmids encoding GST-tagged Pin1, pGEX-KG-Pin1 (wild-type, Y23A, and R68A/R69A mutants) ( , ) and wild-type, Y23A, and R68A/R69A Pin1 in p3xFLAG-14 ( ) were gifts from James Manley (Columbia University).

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: Paragraph title: Expression Constructs ... Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively.

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: For expression in mammalian cells, Daam1 CT fragments were subcloned into pEGFP-C1 (Clontech). .. For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare).

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: Paragraph title: Expression and purification of streptococcal antigens. ... The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Modification:

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SLO (amino acids 1 to 571) cloned into pET-15b ( ) was modified by site-directed mutagenesis to incorporate P427L and W535A mutations. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Western Blot:

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences). .. The whole-cell extract from the induced cells was used for Western-blot analysis.

Transformation Assay:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Derivative Assay:

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: All subclones of the sMMO operon and flanking sequences were derived from pHM2 ( ) (Table ). .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI.

Countercurrent Chromatography:

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The cDNA containing the entire open reading frame was amplified by PCR with the following gene specific primers: ORF-F (5′-CGG ATC CCC TAG AAA AGT GTT CGT T) and ORF-R (5′-CGA ATT CTC ACA GTT TGG AGC GGA TTG T). .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Transfection:

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. For siRNA knockdown, cells were transfected with ON-TARGETplus SMARTpools (GE Healthcare) targeting VPS35 , PDE6D or nontargeting control using DharmaFECT 1 reagent according to the manufacturer’s instructions.

Chromatography:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. Eluted fractions containing GST fusion proteins were further subjected to gel filtration chromatography using a Superdex 200 column (GE Healthcare) with buffer G. Preparation of BAF (non-tagged) and GST-tagged VRK1 by E. coli were performed as described previously ( , ).

Ligation:

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences. .. The retroviral expression vector LZRSpBMN-linker-IRES-EGFP-FLAG-Pin1 (expressing wild-type, Y23A, or R68A/R69A Pin1) was constructed from the respective 3× FLAG Pin1 cDNAs by PCR amplification using primers that introduced a 5′ BglII site and a 3′ XhoI site, followed by ligation of the amplicon into BamHI- and XhoI-digested vector.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SCPA (amino acids 40 to 1039, D130A S512A) sequence was cloned into the pET151d vector by sequence- and ligation-independent cloning (SLIC) using synthetic double-stranded DNA (dsDNA) (gBlock; Integrated DNA Technologies) for the 5′ and 3′ sequences and a PCR-amplified internal sequence from SF30 genomic DNA. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Generated:

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

Sequencing:

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: LZRSpBMN-linker-IRES-EGFP-LSF was constructed by inserting the LSF cDNA sequence from pCMV-LSF in between the BamHI and XhoI sites of the vector ; the S291A, S309A, and the S291A/S309A LSF mutants in the LZRSpBMN-linker-IRES-EGFP background were constructed by a fragment exchange from the appropriate pCMV-LSF construct. .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: Nucleotide substitutions that do not change the amino acid sequence were introduced at the #1 and #2 siRNA target sites, respectively. .. For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare).

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SCPA (amino acids 40 to 1039, D130A S512A) sequence was cloned into the pET151d vector by sequence- and ligation-independent cloning (SLIC) using synthetic double-stranded DNA (dsDNA) (gBlock; Integrated DNA Technologies) for the 5′ and 3′ sequences and a PCR-amplified internal sequence from SF30 genomic DNA. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: The cloned PCR product contained none of the sMMO structural genes; therefore, verification of its sequence was unnecessary. .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI.

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. All plasmids were verified by bidirectional sequencing.

Recombinant:

Article Title: M-Like Proteins of Streptococcus dysgalactiae
Article Snippet: E. coli DH5α [ supE44 ΔlacU169 (φ80 lacZΔM15 ) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ] was used for the expression of recombinant proteins. .. For additional cloning, the plasmid vectors pUC19 and pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) were used.

Article Title: Successful expression and purification of DPPD using a codon optimized synthetic gene *
Article Snippet: If on one hand we were successful to generate a purified recombinant molecule, on the other hand it introduced an undesired property to the rDPPD i.e. , a fusion protein that is no longer specific for M. tuberculosis. .. These included various vectors such pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-2T (GE Healthcare, Piscataway, New Jersey).

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: Paragraph title: Recombinant SCPx protein expression ... The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: Paragraph title: Constructs, Recombinant Proteins, Antibodies, and Other Materials ... For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare).

Cellular Antioxidant Activity Assay:

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: To produce the recombinant SCP2 domain of SCPx protein, the corresponding cDNA region (positions 1381–1671; Figure A below) was amplified by PCR with the following gene-specific primers: SCP2F (5′-GGG ATC CCC GGG CAT CTA CGG ATT CAA AGT) and SCP2R (5′-GGA ATT CGT GAT GGT GAT GGT GAT GCA GTT TGG AGC GGA TTG T). .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Mutagenesis:

Article Title: A novel splicing regulator shares a nuclear import pathway with SR proteins
Article Snippet: The mRRM mutant contained four mutations (Y37A, F39A, Y113A and F115A) in the RRM domain, whereas AAHC had two mutations, C162A and C165A, in the zinc finger. .. The transportin cDNA (from J.Steitz, Yale University, New Haven, CT) was amplified by PCR and inserted in-frame into pGEX-2T (Amersham Pharmacia Biotech).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: All other LSF substitution mutant cDNAs were cloned into the retroviral background by amplifying mutant LSF cDNA from the respective pCMV-LSF, digesting the amplicon with BamHI and XhoI and ligating it into BamHI- and XhoI-digested pLZRSpBMN-linker-IRES-EGFP. .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SLO (amino acids 1 to 571) cloned into pET-15b ( ) was modified by site-directed mutagenesis to incorporate P427L and W535A mutations. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title:
Article Snippet: To generate the GST-DYRK1A/S520A–expressing plasmid, a SacI–NotI fragment (amino acids 200–754) in pGST-DYRK1A was replaced by the equivalent fragment from pHA-DYRK1A/S520A, which contains the point mutation. .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Article Title: Biochemical and Genetic Analyses Provide Insight Into the Structural and Mechanistic Properties of Actin Filament Disassembly by the Aip1p-Cofilin Complex in Saccharomyces cerevisiae
Article Snippet: Paragraph title: aip1-GST and cof1-GST mutant plasmid construction: ... Vector pBH360 was created by cloning a cofilin PCR product into the Bam HI and Eco RI sites of pGEX-2T (GE Healthcare).

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

Isolation:

Article Title:
Article Snippet: The human 14-3-3β open reading frame was amplified by polymerase chain reaction (PCR) from the pACTII-14-3-3β plasmid isolated from the cDNA library used in the yeast two-hybrid screen (human fetal brain library in pACTII; Clontech, Mountain View, CA). .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Subcloning:

Article Title:
Article Snippet: Plasmid pEGFP-DYRK1A/1–167 was made by subcloning a BamHI–SalI PCR fragment (amino acids 1–167) into pEGFP BglII-XhoI. .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Purification:

Article Title: Successful expression and purification of DPPD using a codon optimized synthetic gene *
Article Snippet: If on one hand we were successful to generate a purified recombinant molecule, on the other hand it introduced an undesired property to the rDPPD i.e. , a fusion protein that is no longer specific for M. tuberculosis. .. These included various vectors such pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-2T (GE Healthcare, Piscataway, New Jersey).

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences). .. The GST (glutathione S-transferase)–SCP2 fusion protein was purified with glutathione–agarose (Sigma).

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare). .. The proteins were purified with glutathione-Sepharose (GE Healthcare) for GST-fused proteins or with Ni2+ -nitrilotriacetic acid-agarose (Qiagen) for His6 -tagged proteins according to the manufacturers' instructions.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: Paragraph title: Expression and purification of streptococcal antigens. ... The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Polymerase Chain Reaction:

Article Title: A novel splicing regulator shares a nuclear import pathway with SR proteins
Article Snippet: .. The transportin cDNA (from J.Steitz, Yale University, New Haven, CT) was amplified by PCR and inserted in-frame into pGEX-2T (Amersham Pharmacia Biotech). ..

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: The resulting PCR product was gel-purified and cloned into pGEM®-T Easy vector. .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences. .. The retroviral expression vector LZRSpBMN-linker-IRES-EGFP-FLAG-Pin1 (expressing wild-type, Y23A, or R68A/R69A Pin1) was constructed from the respective 3× FLAG Pin1 cDNAs by PCR amplification using primers that introduced a 5′ BglII site and a 3′ XhoI site, followed by ligation of the amplicon into BamHI- and XhoI-digested vector.

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. .. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6 -SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SCPA (amino acids 40 to 1039, D130A S512A) sequence was cloned into the pET151d vector by sequence- and ligation-independent cloning (SLIC) using synthetic double-stranded DNA (dsDNA) (gBlock; Integrated DNA Technologies) for the 5′ and 3′ sequences and a PCR-amplified internal sequence from SF30 genomic DNA. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title:
Article Snippet: .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare). .. The anti-DYRK1A antibody used in this study was raised by immunizing rabbits with a purified GST-fusion protein making up the last 144 amino acids of DYRK1A.

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: The cloned PCR product contained none of the sMMO structural genes; therefore, verification of its sequence was unnecessary. .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI.

Article Title: Biochemical and Genetic Analyses Provide Insight Into the Structural and Mechanistic Properties of Actin Filament Disassembly by the Aip1p-Cofilin Complex in Saccharomyces cerevisiae
Article Snippet: .. Vector pBH360 was created by cloning a cofilin PCR product into the Bam HI and Eco RI sites of pGEX-2T (GE Healthcare). ..

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: .. Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Positron Emission Tomography:

Article Title: Successful expression and purification of DPPD using a codon optimized synthetic gene *
Article Snippet: .. These included various vectors such pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-2T (GE Healthcare, Piscataway, New Jersey). .. Host E. coli cells tested included BL21(DE3), BL21(DE3)pLysS, and Rosetta 2(DE)pLysS (Novagen).

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively. .. Full-length X. tropicalis TOCA1, TOCA1 F-BAR (residues 1–287), and TOCA1 ΔSH3 (residues 1–480) were PCR-amplified from a cDNA clone (IMAGE 5157175) and cloned into pET-His6 -SNAP using FseI and AscI sites that had been incorporated into the primers to create His-SNAP-TOCA1 proteins.

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SLO (amino acids 1 to 571) cloned into pET-15b ( ) was modified by site-directed mutagenesis to incorporate P427L and W535A mutations. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title: VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Article Snippet: .. Plasmids and RNA interference Full-length or partial coding regions of each cDNA were amplified by PCR and cloned in-frame into the indicated vectors, including pEGFP-C1, pmCherry-C1, pEYFP-N1, pECFP-C1 (Takara Bio Inc.), pcDNA5/FRT/TO, pcDNA3.1+ (Invitrogen), pTRIPz (GE Healthcare), pGEX-2T (GE Healthcare), pET-14b (EMD Millipore), and pCGN ( ). .. Amino acid substitutions were generated by site-directed mutagenesis using the QuikChange XL Site-Directed Mutagenesis kit (Agilent Technologies).

cDNA Library Assay:

Article Title: Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay *
Article Snippet: .. ORFs of RFPL3, RNF25, STUB1, and TRIM52 were amplified from a cDNA library and cloned into the SmaI site of pGEX-2T (GE Healthcare). .. The pGEX-2T constructs were transformed into the E. coli BL21(DE3) strain (Stratagene), and protein expression was induced as described previously ( ).

Article Title: A novel splicing regulator shares a nuclear import pathway with SR proteins
Article Snippet: Moreover, the Lark cDNA was amplified by PCR using a Drosophila embryonic cDNA library (gift of H.Sun, Academia Sinica, Taipei, Taiwan). .. The transportin cDNA (from J.Steitz, Yale University, New Haven, CT) was amplified by PCR and inserted in-frame into pGEX-2T (Amersham Pharmacia Biotech).

Article Title:
Article Snippet: The human 14-3-3β open reading frame was amplified by polymerase chain reaction (PCR) from the pACTII-14-3-3β plasmid isolated from the cDNA library used in the yeast two-hybrid screen (human fetal brain library in pACTII; Clontech, Mountain View, CA). .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Chloramphenicol Acetyltransferase Assay:

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: To produce the recombinant SCP2 domain of SCPx protein, the corresponding cDNA region (positions 1381–1671; Figure A below) was amplified by PCR with the following gene-specific primers: SCP2F (5′-GGG ATC CCC GGG CAT CTA CGG ATT CAA AGT) and SCP2R (5′-GGA ATT CGT GAT GGT GAT GGT GAT GCA GTT TGG AGC GGA TTG T). .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Plasmid Preparation:

Article Title: M-Like Proteins of Streptococcus dysgalactiae
Article Snippet: .. For additional cloning, the plasmid vectors pUC19 and pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden) were used. .. Restriction endonucleases and Taq DNA polymerase were either from Amersham Pharmacia Biotech or MBI Fermentas (Vilnius, Lithuania).

Article Title: Successful expression and purification of DPPD using a codon optimized synthetic gene *
Article Snippet: These included various vectors such pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-2T (GE Healthcare, Piscataway, New Jersey). .. However, as mentioned before, low levels of rDPPD could be expressed and purified by using the Mycobacterium expression vector pSMT3 and Mycobacterium smegmatis as host cell [ ].

Article Title: A novel splicing regulator shares a nuclear import pathway with SR proteins
Article Snippet: Paragraph title: Plasmid constructions ... The transportin cDNA (from J.Steitz, Yale University, New Haven, CT) was amplified by PCR and inserted in-frame into pGEX-2T (Amersham Pharmacia Biotech).

Article Title: Characterization of a sterol carrier protein 2/3-oxoacyl-CoA thiolase from the cotton leafworm (Spodoptera littoralis): a lepidopteran mechanism closer to that in mammals than that in dipterans
Article Snippet: .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences). .. The recombinant protein was expressed using the conditions recommended by the manufacturer.

Article Title: Prolyl Isomerase Pin1 Regulates Transcription Factor LSF (TFCP2) by Facilitating Dephosphorylation at Two Serine-Proline Motifs *
Article Snippet: .. The bacterial expression plasmid encoding glutathione S -transferase (GST), pGEX-2T was from Amersham Biosciences. .. Bacterial expression plasmids encoding GST-tagged Pin1, pGEX-KG-Pin1 (wild-type, Y23A, and R68A/R69A mutants) ( , ) and wild-type, Y23A, and R68A/R69A Pin1 in p3xFLAG-14 ( ) were gifts from James Manley (Columbia University).

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: SCPA (amino acids 40 to 1039, D130A S512A) sequence was cloned into the pET151d vector by sequence- and ligation-independent cloning (SLIC) using synthetic double-stranded DNA (dsDNA) (gBlock; Integrated DNA Technologies) for the 5′ and 3′ sequences and a PCR-amplified internal sequence from SF30 genomic DNA. .. The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ).

Article Title:
Article Snippet: The human 14-3-3β open reading frame was amplified by polymerase chain reaction (PCR) from the pACTII-14-3-3β plasmid isolated from the cDNA library used in the yeast two-hybrid screen (human fetal brain library in pACTII; Clontech, Mountain View, CA). .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Article Title: Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase
Article Snippet: .. -RK2) from plasmid RK2 ( pNPB101, the subclone containing the 5′ portion of mmoX , which was used as the target DNA for mutagenesis, was constructed by cloning the smaller (1.0-kb) Xba I- Nde I fragment of pSJH1a (Fig. ) into pUC18. pSJH2, the construct used for the expression of the glutathione S -transferase (GST)-protein B fusion, was constructed by amplifying the mmoB gene from pSJH1a with primers 5′-G ATC GGA TCC ATG TCC AGC GCT CAT AAC G-3′ ( Bam HI site is underlined) and 5′-G ATC GAA TTC CGA TCA GAT GTC GGT CAG-3′ ( Eco RI site is underlined) and cloning it into pGEX-2T (Amersham-Pharmacia) by using Bam HI and Eco RI. .. The mutagenesis of mmoX was performed by means of the four-primer overlap extension PCR method ( ) with pNPB101 as the template.

Article Title: Biochemical and Genetic Analyses Provide Insight Into the Structural and Mechanistic Properties of Actin Filament Disassembly by the Aip1p-Cofilin Complex in Saccharomyces cerevisiae
Article Snippet: .. Vector pBH360 was created by cloning a cofilin PCR product into the Bam HI and Eco RI sites of pGEX-2T (GE Healthcare). ..

Article Title: Biochemical and Structural Definition of the l-Afadin- and Actin-binding Sites of α-Catenin *
Article Snippet: .. Full-length α -catenin used in the limited proteolysis experiment was expressed with a C-terminal His6 -tag ( ); for expression of the N-terminal GST fusion protein, full-length α -catenin was inserted into the pGEX-KG vector, a modified form of pGEX-2T (Amersham Biosciences), in which a linker of 5 glycine residues is introduced between the thrombin cleavage and the multiple cloning sites ( ).2 All α -catenin fragments were amplified by PCR. α -Catenin 385–651 and α -catenin 385–507 were cloned into the pGEX-2T expression vector (Amersham Biosciences). α -Catenin 632–906, α -catenin 671–906, and α -catenin 678 – 864 were cloned into the pGEX-4T-3 vector (Amersham Biosciences), and α -catenin 507–632 was introduced into the pGEX-KG vector. ..

Affinity Chromatography:

Article Title: Differing Efficacies of Lead Group A Streptococcal Vaccine Candidates and Full-Length M Protein in Cutaneous and Invasive Disease Models
Article Snippet: The gene encoding M1 protein (amino acids 13 to 455) was cloned into pGEX-2T (GE Healthcare Life Sciences), incorporating a carboxy-terminal 6×His tag ( ). .. ADI, TF, SpyCEP, SLO, and SCPA antigens were expressed in E. coli BL21 Star (DE3) cells and purified by immobilized metal ion affinity chromatography (IMAC).

Two Hybrid Screening:

Article Title:
Article Snippet: The human 14-3-3β open reading frame was amplified by polymerase chain reaction (PCR) from the pACTII-14-3-3β plasmid isolated from the cDNA library used in the yeast two-hybrid screen (human fetal brain library in pACTII; Clontech, Mountain View, CA). .. The PCR product was cloned into pGEM-Teasy (Promega, Madison, WI), sequenced, and then inserted in-frame into the BamHI and EcoRI sites of pGEX-2T (GE Healthcare).

Protein Binding:

Article Title: Investigation of the Interaction between Cdc42 and Its Effector TOCA1
Article Snippet: The HR1 domain of human CIP4 (residues 388–481) was amplified from IMAGE clone 3532036, the Xenopus laevis FBP17 HR1 domain (residues 385–486) from IMAGE clone 5514481, and the X. tropicalis N-WASP G protein-binding domain (GBD) (residues 197–255) from IMAGE clone 5379332, and all were cloned into pGEX-6P-1. .. Human Cdc42Δ7Q61L and full-length Cdc42 were cloned into pGEX-2T (GE Healthcare) and pGEX-6P-1, respectively.

Produced:

Article Title: Flightless-I (Fli-I) Regulates the Actin Assembly Activity of Diaphanous-related Formins (DRFs) Daam1 and mDia1 in Cooperation with Active Rho GTPase *
Article Snippet: Full-length Fli-I, which is insensitive to siRNA against mouse Fli-I, was produced by PCR using human Fli-I as a template. .. For generation of glutathione S -transferase (GST)-fused proteins, gene fragments were subcloned into pGEX-2T (GE Healthcare).

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    GE Healthcare gst
    Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) <t>GST–syndapin</t> I, II, and III specifically precipitate <t>GFP-ProSAP1</t> expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).
    Gst, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    GE Healthcare gst syndapin i sh3 domain
    <t>Syndapin</t> I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both <t>SH3</t> domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P
    Gst Syndapin I Sh3 Domain, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare expression vector pgex 2t
    Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector <t>pGEX-2T.</t> b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences
    Expression Vector Pgex 2t, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GE Healthcare gst vector control
    <t>Drp1</t> binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed <t>GST,</t> GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P
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    Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Binding Assay, Mutagenesis, In Vitro, Purification

    Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: In Vivo, Immunolabeling, Mouse Assay, Transfection, Staining, Marker

    Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Syndapin I depletion reduces the frequencies of mEPSCs. (A and F) Sample traces of whole-cell patch clamp recordings of mEPSCs from individual primary rat hippocampal neurons transfected at DIV 12 and analyzed 48 h later. (B) The frequency of mEPSCs was reduced in syndapin I RNAi neurons when compared with pRNAT and scrambled RNAi, respectively (B and D), whereas the mEPSC amplitudes did not differ (C and E). (F–J) Syndapin I RNAi rescue experiments with coexpression of mCherry–syndapin I and mutants thereof showing that both SH3 domain protein interactions and F-BAR domain–mediated membrane interactions are crucial for syndapin I functions in postsynaptic neurotransmission. *, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Patch Clamp, Transfection

    Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Impaired spine and synapse formation upon syndapin I loss-of-function is caused by a loss of SH3 domain-dependent syndapin I functions in the postsynaptic compartment. (A and H) PM-mCherry signals of dendrites of neurons transfected as indicated at DIV 12 and fixed at DIV 14. Bars, 5 µm. (B–D) Quantitative analyses of general spine density (B) and of individual morphology groups (C and D) upon syndapin I RNAi. (E) Anti–PSD-95 (postsynaptic) and anti–synapsin 1 (presynaptic) immunolabeling along dendrites of transfected neurons. Bar, 5 µm. (F and G) Quantitation of PSD-95– (F) and synapsin 1–positive puncta (G) spatially overlapping with transfected neurons. (I–K) Quantitative analyses of general spine density (I) and of individual morphology groups (J and K) of syndapin I–depleted cells expressing Sdp I ΔSH3 compared with pRNAT control cells transfected in parallel. **, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Transfection, Immunolabeling, Quantitation Assay, Expressing

    ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: ProSAP1-mediated functions in spine head enlargement rely on complex formation with syndapin I. (A–G) Absence of ProSAP1-mediated spine head enlargement upon coexpression of the syndapin I SH3 domain blocking the syndapin I binding site of ProSAP1 (A and B), upon use of ProSAP1* (C–E), and upon concomitant syndapin I RNAi (F and G), respectively. (A, D, and F) Representative images of neurons transfected as indicated (cotransfected with PM-mCherry for morphological analysis). (B, E, and G) Quantification of head width of mushroom spines. (C) Coprecipitation studies with immobilized syndapin I and Abp1 SH3 domains and GFP-ProSAP1 versus GFP-ProSAP1* showing specific disruption of syndapin I interaction. (H–J) Quantitative analysis of head width of mushroom spines. Neither syndapin I RNAi nor overexpression of syndapin I modulate head sizes of mushroom spines. (J) ProSAP RNAi causes a decrease in head width not seen upon syndapin I RNAi and not rescued by syndapin I coexpression. *, P

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Blocking Assay, Binding Assay, Transfection, Over Expression

    Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Identification of ProSAP1/Shank2 and ProSAP2/Shank3 as postsynaptically enriched Syndapin I interaction partners. (A) GST–syndapin I, II, and III specifically precipitate GFP-ProSAP1 expressed in HEK293 cells. (B) Coprecipitation analysis with GST–syndapin I and deletion mutants thereof. The SH3 domain is critical and sufficient for binding. A mutant SH3 domain (P434L; SH3*) did not bind. White lines indicate lanes omitted from blots (B and F). (C) Syndapin I SH3 precipitates GFP-ProSAP1 and GFP-ProSAP2 but not GFP-Shank1. (D) Alignment of +++APPPP motifs in ProSAP1 (NCBI Protein database accession no. NP_001004133 ), ProSAP2 (accession no. NP_067708 ), and Cobl (accession no. NP_766084 ; conserved amino acids are highlighted) and of corresponding residues in Shank1 (accession no. Q9WV48 ). (E) Scheme of rat ProSAP1b and deletion mutants used. Indicated are the N-terminal PDZ domain (medium grey), several proline-rich motifs (dark grey lines), and the C-terminal SAM (sterile alpha motif) domain (light grey). (F) GST–syndapin I precipitated GFP-ProSAP1 1–235 but none of the other ProSAP1 deletion mutants. (G) GFP fusion peptides encompassing the +++APPPP motifs of ProSAP1, ProSAP2, and Cobl associated with syndapin I SH3. (H and I) RKKAPPPPKR to GAGAAAAAAG mutation (amino acids 141–150 in ProSAP1; ProSAP1 1–235*) disrupted direct binding of ProSAP1 to syndapin I in both in vitro reconstitutions with purified proteins (H) and in coprecipitation analyses (I).

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: Binding Assay, Mutagenesis, In Vitro, Purification

    Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Journal: The Journal of Cell Biology

    Article Title: ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

    doi: 10.1083/jcb.201307088

    Figure Lengend Snippet: Syndapin I interacts with ProSAP1 in vivo. (A) Specific coimmunoprecipitation of GFP-ProSAP1 with anti-FLAG antibodies immunoprecipitating FLAG–syndapin I. (B) Consistently, FLAG–syndapin I (arrowhead) was specifically coimmunoprecipitated with GFP-ProSAP1. (C) Immobilized GST–syndapin I SH3 specifically precipitated endogenous ProSAP1 from mouse brain cytosol (MBC). (D) Endogenous syndapin I was precipitated from rat brain preparations (RBC) with immobilized GST-ProSAP1 139–153 comprising the RKKAPPPP motif. (E–H) Syndapin I constitutively targeted to outer mitochondrial membranes recruited GFP-ProSAP1 (E) and GFP-ProSAP1 1–235 (F) in intact COS-7 cells, whereas Sdp I ΔSH3 did not (G and H). Bars, 10 µm. (I) Syndapin I and ProSAP1 immunolabeling of brain sections from adult mice. Colocalization in synapses of mossy fibers with dendrites of pyramidal cells in the stratum lucidum in the hippocampus CA3 is shown. Blue signal in merge, DAPI. Insets, 2.5-fold enlargements of the boxed areas. Bars, 25 µm. (J) Immunolabeling of neurons transfected with Xpress–syndapin I at DIV 12 and stained for syndapin I, ProSAP1, and the dendritic marker MAP2 at DIV 14. Insets, 1.5-fold enlargements of boxed areas. Bar, 10 µm. (K) Endogenous syndapin I colocalized with ProSAP1 and synapsin 1 (DIV 21). Insets, twofold enlargements of boxed areas. Bars: (main panels) 5 µm; (insets) 2 µm.

    Article Snippet: DNA constructs Plasmids encoding for GFP (pEGFP-C1; Takara Bio Inc.)-, GST (pGEX-2T; GE Healthcare)-, Xpress (pcDNA 3.1/HisC; Invitrogen)-, and FLAG-tagged (pCMV-Tag2b; Agilent Technologies) full-length syndapin I as well as for GST–syndapin I SH3 domain (aa 376–441, pGEX-2T; aa 378–441, pGEX-5X-1; GE Healthcare), GST–syndapin I SH3P434L (GST-Sdp I SH3*; aa 376–441, pGEX-2T), and GST–syndapin IΔSH3 (aa 1–382, pGEX-2T) were described in , , and , respectively.

    Techniques: In Vivo, Immunolabeling, Mouse Assay, Transfection, Staining, Marker

    Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences

    Journal: Inflammation Research

    Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies

    doi: 10.1007/s00011-016-0987-1

    Figure Lengend Snippet: Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences

    Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria).

    Techniques: Recombinant, HMT Assay, Clone Assay, Expressing, Plasmid Preparation, Staining, Purification, Mouse Assay, Molecular Weight, Sequencing, Produced

    Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P

    Journal: The Journal of Clinical Investigation

    Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

    doi: 10.1172/JCI127277

    Figure Lengend Snippet: Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P

    Article Snippet: Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h).

    Techniques: Cell Culture, Plasmid Preparation, Transfection, Purification, GST Pulldown Assay, Staining, SDS Page, Binding Assay, Mutagenesis, shRNA, Expressing, Stable Transfection, Sampling