Structured Review

GE Healthcare pgex 2t
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 <t>pGEX-2T</t> 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.
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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:

doi: 10.1042/BJ20040717

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 ""

Article Title:

Journal:

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:

doi: 10.1042/BJ20040717

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 "Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly"

Article Title: Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly

Journal:

doi: 10.1091/mbc.E04-02-0121

Inhibition of microtubule nucleation from the purified centrosome. (A) cDNA constructs containing various portions of CPAP were subcloned into the pGEX-2T vector and expressed as GST-CPAP–truncated proteins in E. coli . (B) Coomassie Blue-stained gel with various affinity-purified GST-CPAP–truncated proteins used in microtubule nucleation assay. GST-PN1 (lane 1), GST-PN2 (lane 2), GST-A5N (lane 3), GST-CM (lane 4), GST-A5C (lane5), GST-A5M2-1 (lane 6). GST-A5M2-3 (lane7), GST-A5M2-2 (lane 8), and bovine serum albumin (BSA) (lane 9, 1 μg). (C) In vitro microtubule nucleation assay. The isolated centrosomes were preincubated with various GST-CPAP–truncated proteins (0.15 μM) for 10 min at 4°C, and then tubulin was added to initiate the microtubule nucleation. The microtubule asters were stained with FITC-conjugated anti-α-tubulin antibodies (DM1A-FITC). Bar, 10 μm. Five α-helical coiled-coil structures (I–V) and a G-box (glycine repeats) are indicated ( <xref ref-type= Hung et al ., 2000 ). " title="... various portions of CPAP were subcloned into the pGEX-2T vector and expressed as GST-CPAP–truncated proteins in E. ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Inhibition of microtubule nucleation from the purified centrosome. (A) cDNA constructs containing various portions of CPAP were subcloned into the pGEX-2T vector and expressed as GST-CPAP–truncated proteins in E. coli . (B) Coomassie Blue-stained gel with various affinity-purified GST-CPAP–truncated proteins used in microtubule nucleation assay. GST-PN1 (lane 1), GST-PN2 (lane 2), GST-A5N (lane 3), GST-CM (lane 4), GST-A5C (lane5), GST-A5M2-1 (lane 6). GST-A5M2-3 (lane7), GST-A5M2-2 (lane 8), and bovine serum albumin (BSA) (lane 9, 1 μg). (C) In vitro microtubule nucleation assay. The isolated centrosomes were preincubated with various GST-CPAP–truncated proteins (0.15 μM) for 10 min at 4°C, and then tubulin was added to initiate the microtubule nucleation. The microtubule asters were stained with FITC-conjugated anti-α-tubulin antibodies (DM1A-FITC). Bar, 10 μm. Five α-helical coiled-coil structures (I–V) and a G-box (glycine repeats) are indicated ( Hung et al ., 2000 ).

Techniques Used: Inhibition, Purification, Construct, Plasmid Preparation, Staining, Affinity Purification, In Vitro, Isolation, Electron Microscopy

Mapping the microtubule-destabilizing activity of CPAP to the PN2-3 motif. (A) Three PN2 subregions, PN2-1, PN2-2, and PN2-3, were subcloned into a pGEX-2T vector. (B) Various GST-recombinant proteins, GST-PN2-1 (lane 1), GST-PN2-2 (lane 2), and GST-PN2-3 (lane 3), were affinity purified and stained with Coomassie Blue. Lane 4 represents 1 μg of bovine serum albumin (BSA). (C and D) In vitro microtubule nucleation assay. Various GST-CPAP–truncated proteins (0.45 μM) were preincubated with purified centrosomes as described in . The microtubule asters were stained with FITC-conjugated anti-α-tubulin antibodies (green color) (C), or doubly stained with both anti-α-tubulin monoclonal antibodies (green) and anti-GST polyclonal antibodies (red) (D). The inserted inlet (a) in Figure 2D is a merged photograph derived from two monochrome photos, b (anti-GST antibody, red) and c (anti-α-tubulin antibody, green). Bar, 10 μm.
Figure Legend Snippet: Mapping the microtubule-destabilizing activity of CPAP to the PN2-3 motif. (A) Three PN2 subregions, PN2-1, PN2-2, and PN2-3, were subcloned into a pGEX-2T vector. (B) Various GST-recombinant proteins, GST-PN2-1 (lane 1), GST-PN2-2 (lane 2), and GST-PN2-3 (lane 3), were affinity purified and stained with Coomassie Blue. Lane 4 represents 1 μg of bovine serum albumin (BSA). (C and D) In vitro microtubule nucleation assay. Various GST-CPAP–truncated proteins (0.45 μM) were preincubated with purified centrosomes as described in . The microtubule asters were stained with FITC-conjugated anti-α-tubulin antibodies (green color) (C), or doubly stained with both anti-α-tubulin monoclonal antibodies (green) and anti-GST polyclonal antibodies (red) (D). The inserted inlet (a) in Figure 2D is a merged photograph derived from two monochrome photos, b (anti-GST antibody, red) and c (anti-α-tubulin antibody, green). Bar, 10 μm.

Techniques Used: Activity Assay, Plasmid Preparation, Recombinant, Affinity Purification, Staining, In Vitro, Purification, Derivative Assay

Related Articles

Clone Assay:

Article Title:
Article Snippet: A GST-tagged barrier to autointegration factor (GST-BAF) and vaccinia-related kinase 1 (GST-VRK1) were also produced by the same split-primer PCR method using antisense primers, AODS (for the first PCR), and AODS-3 (for the second PCR) (CellFree Sciences) and a control protein, GST-tagged bacterial dihydrofolate reductase (GST-DHFR), was produced using AODA2306 and AODA2303 from the plasmid DNAs described previously ( , , ). .. 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).

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Article Title: Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region
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Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
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Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
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Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
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Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: Paragraph title: Cloning and Purification ... The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct.

Centrifugation:

Article Title:
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: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The cells were harvested by centrifugation at 4°C and washed with cold phosphate-buffered saline (PBS) before being processed for cell lysis, SDS-PAGE, and immunoblotting. .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ).

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. BL21 DE3 pLysS E. coli were transformed with pGEX-2T-Dlg1 PDZ2, grown in LB media under 50 µg/L ampicillin selection at 37°C to an optical density at 600 nm of 1.0 at which point protein expression was induced by the addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (final concentration), the temperature was lowered to 20°C, and induction proceeded for 16 hours.

Amplification:

Article Title:
Article Snippet: A GST-tagged barrier to autointegration factor (GST-BAF) and vaccinia-related kinase 1 (GST-VRK1) were also produced by the same split-primer PCR method using antisense primers, AODS (for the first PCR), and AODS-3 (for the second PCR) (CellFree Sciences) and a control protein, GST-tagged bacterial dihydrofolate reductase (GST-DHFR), was produced using AODA2306 and AODA2303 from the plasmid DNAs described previously ( , , ). .. 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: 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: UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration
Article Snippet: A portion of cDNA encoding BICD-1(227–737) was amplified by PCR with the appropriate restriction sites and cloned into the Bam HI site of pGEX-2T to create a GST-BICD-1 fusion protein (pSL294). .. Full-length nud-2 cDNA was amplified by PCR with the appropriate overhanging restriction sites and cloned into the Pvu I and Sac I sites of pGEX-2T to create a GST-NUD-2 fusion protein (pSL246). cDNA encoding NUD-2(239–293) was amplified by PCR with MfeI and BamHI overhanging restriction sites and cloned into the EcoRI and BamHI sites of pGEX-2T to create a GST-NUD-2(239–293) fusion protein (pSL424). .. Full-length egal-1 cDNA was amplified by PCR with the appropriate overhanging restriction sites and cloned into the Bam HI and Hind III sites of pMAL-c2 (New England Biolabs, Ipswich, MA) to create an MBP-EGAL-1 fusion protein (pSL396). cDNA enoding residues 1–698 of UNC-83c were used in the MBP-UNC-83 fusion construct (pDS31; ( ).

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. GST-NBL1 protein was purified as described previously [ , ].

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. GST-NBL1 protein was purified as described previously [ , ].

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Various Gal4-DBD-TR fusion proteins have been designed by PCR amplification of chicken TRα fragments which were cloned into pcDNA3 (Life Technologies, Darmstadt, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany). .. Point mutations were introduced using a self-made site-directed mutagenesis system adapted from the QuickChange kit by Stratagene.

Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: The Pf tRip gene was amplified by PCR from a P. falciparum cDNA library (provided by H. Vial, CNRS UMR 5235, University of Montpellier 2, Montpellier, France) and sequenced. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein.

Filtration:

Article Title:
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.

Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein.

TA Cloning:

Article Title: β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms
Article Snippet: E. coli strains were cultured in LB-Miller medium or agar plate (BD Difco, Franklin Lakes, NJ) with appropriate antibiotics. .. For gene cloning, pGEM-T TA cloning vector (Promega, Madison, WI) was employed and pGEX-2T (GE Healthcare Life Science, Pittsburgh, PA) was used for recombinant protein expression. .. Fold change in gene expression in cobiofilms versus single species was determined by reverse transcription-quantitative PCR (qRT-PCR) using the double delta cycle threshold (ΔΔ CT ) method as described previously ( ).

Construct:

Article Title: The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
Article Snippet: Antisera against Francisella Lon, ClpP, ClpX, and FTL1017 were prepared with recombinant forms of these proteins in New Zealand White rabbits essentially as described previously ( ). .. Lon, ClpP, and ClpX were expressed as glutathione S -transferase (GST) fusion proteins by amplifying their coding regions from LVS genomic DNA with primer pairs Pr3230/Pr3231 ( lon ), Pr5476/Pr5477 ( clpP ), and Pr5478/Pr5479 ( clpX ) and cloning each of the PCR products in the NotI/AscI site of pST2700, a modified pGEX-2T with additional cloning sites ( ). pST2700 was constructed by insertion of an MCS sequence into the BamHI/EcoRI site of pGEX-2T. .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region
Article Snippet: Paragraph title: Protein expression constructs ... All WASP–GBD peptides were expressed as GST fusion proteins in pGEX-2T (Amersham Biosciences) with residues 201–321 of WASP cloned into BamH1 and EcoR1 sites of the multicloning site ( ).

Article Title: The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP
Article Snippet: Escherichia coli expression plasmids for GST, GST-HAT (1096–1757 amino acids) and GST-C/H3 (1514–1894 amino acids) have been described ( ). .. GST-N-HAT (1096–1514 amino acids), GST-C-HAT (1514–1679 amino acids), GST-ΔC-HAT (1514–1723 amino acids) and GST-TAZ2 (1758–1894 amino acids) were constructed by PCR-amplification of pRc/RSV-CBP ( ) and cloning products into pGEX-2T (GE Healthcare) at the BamHI/EcoRI sites. .. GST-HBZ and GST-HBZ-Mut(LXXAA)2 , HBZ-ΔbZIP (1–122 amino acids) and HBZ-bZIP(120–206 amino acids) , pRSETA-p53 ( ) have been described. c-Jun bZIP (257–334 amino acids) was PCR-amplified from pBiFC-bJunVN173 ( ) and cloned into pRSETA at the BamHI/EcoRI sites.

Article Title: A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms
Article Snippet: 2.1 The β-glucuronidase gene of pBI121 was replaced by the insertion of the cDNA fragments encoding SoCSaseA and SoCSaseLP to produce the overexpression constructs, pBI-CSaseA and pBI-CSaseLP, respectively. .. For expression of a fusion protein with glutathione S -transferase (GST), the SoCSaseLP cDNA fragment was inserted into the Bam HI/Eco RI site of pGEX-2T (GE Healthcare Japan, Tokyo, Japan).

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Cdk8 siRNA (Cell Signaling Technology, Danvers, MA) was introduced into cells using siPORT transfection reagent (Invitrogen) according to the manufacturer's instructions. .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA). .. The fusion proteins, as well as the GST control, were expressed and purified from E. coli as suggested by the manufacturer.

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: DNA encoding human Dlg1 PDZ2, residues 310–407, was generated by PCR sewing using oligonucleotide templates with optimal E. coli codon usage. .. The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. BL21 DE3 pLysS E. coli were transformed with pGEX-2T-Dlg1 PDZ2, grown in LB media under 50 µg/L ampicillin selection at 37°C to an optical density at 600 nm of 1.0 at which point protein expression was induced by the addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (final concentration), the temperature was lowered to 20°C, and induction proceeded for 16 hours.

Incubation:

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. Amplified luminescence proximity homogeneous assay (AlphaLISA) was performed in 384-well microtiter plates (white opaque ProxiPlate™ , PerkinElmer, Waltham, MA, USA) containing 2.5 μL of 1/100-diluted sera and 2.5 μL of GST or a GST-fusion protein (10 μg/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500 and 0.05% Proclin-300).

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. Amplified luminescence proximity homogeneous assay (AlphaLISA) was performed in 384-well microtiter plates (white opaque ProxiPlate™ , PerkinElmer, Waltham, MA, USA) containing 2.5 μL of 1/100-diluted sera and 2.5 μL of GST or a GST-fusion protein (10 μg/mL) in AlphaLISA buffer (25 mM HEPES, pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL dextran-500 and 0.05% Proclin-300).

In Silico:

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany).

Expressing:

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: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region
Article Snippet: Paragraph title: Protein expression constructs ... All WASP–GBD peptides were expressed as GST fusion proteins in pGEX-2T (Amersham Biosciences) with residues 201–321 of WASP cloned into BamH1 and EcoR1 sites of the multicloning site ( ).

Article Title: The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP
Article Snippet: Escherichia coli expression plasmids for GST, GST-HAT (1096–1757 amino acids) and GST-C/H3 (1514–1894 amino acids) have been described ( ). .. GST-N-HAT (1096–1514 amino acids), GST-C-HAT (1514–1679 amino acids), GST-ΔC-HAT (1514–1723 amino acids) and GST-TAZ2 (1758–1894 amino acids) were constructed by PCR-amplification of pRc/RSV-CBP ( ) and cloning products into pGEX-2T (GE Healthcare) at the BamHI/EcoRI sites.

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: The expression plasmid of human TRβ1 is a kind gift from Peter Hofmann (Charité Berlin, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany).

Article Title: A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms
Article Snippet: 2.1 The β-glucuronidase gene of pBI121 was replaced by the insertion of the cDNA fragments encoding SoCSaseA and SoCSaseLP to produce the overexpression constructs, pBI-CSaseA and pBI-CSaseLP, respectively. .. For expression of a fusion protein with glutathione S -transferase (GST), the SoCSaseLP cDNA fragment was inserted into the Bam HI/Eco RI site of pGEX-2T (GE Healthcare Japan, Tokyo, Japan). .. The resultant plasmid, pGST-CSLP, was used for production of a GST-fused SoCSaseLP (called GST-CSLP).

Article Title: β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms
Article Snippet: E. coli strains were cultured in LB-Miller medium or agar plate (BD Difco, Franklin Lakes, NJ) with appropriate antibiotics. .. For gene cloning, pGEM-T TA cloning vector (Promega, Madison, WI) was employed and pGEX-2T (GE Healthcare Life Science, Pittsburgh, PA) was used for recombinant protein expression. .. Fold change in gene expression in cobiofilms versus single species was determined by reverse transcription-quantitative PCR (qRT-PCR) using the double delta cycle threshold (ΔΔ CT ) method as described previously ( ).

BIA-KA:

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The resulting plasmids pTH2604 (PtvB) and pTH2760 (SpxB) were used to produce recombinant proteins by affinity chromatography with glutathione-Sepharose 4 Fast Flow resins (GE Healthcare BioSciences) according to the supplier's instructions.

Modification:

Article Title: The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
Article Snippet: Antisera against Francisella Lon, ClpP, ClpX, and FTL1017 were prepared with recombinant forms of these proteins in New Zealand White rabbits essentially as described previously ( ). .. Lon, ClpP, and ClpX were expressed as glutathione S -transferase (GST) fusion proteins by amplifying their coding regions from LVS genomic DNA with primer pairs Pr3230/Pr3231 ( lon ), Pr5476/Pr5477 ( clpP ), and Pr5478/Pr5479 ( clpX ) and cloning each of the PCR products in the NotI/AscI site of pST2700, a modified pGEX-2T with additional cloning sites ( ). pST2700 was constructed by insertion of an MCS sequence into the BamHI/EcoRI site of pGEX-2T. .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

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 recombinant protein was expressed using the conditions recommended by the manufacturer.

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA). .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: Protein bands were visualized by the Clarity Western enhanced chemiluminescence reagent (Bio-Rad) according to the supplier's instructions. .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ).

Over Expression:

Article Title: A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms
Article Snippet: 2.1 The β-glucuronidase gene of pBI121 was replaced by the insertion of the cDNA fragments encoding SoCSaseA and SoCSaseLP to produce the overexpression constructs, pBI-CSaseA and pBI-CSaseLP, respectively. .. For expression of a fusion protein with glutathione S -transferase (GST), the SoCSaseLP cDNA fragment was inserted into the Bam HI/Eco RI site of pGEX-2T (GE Healthcare Japan, Tokyo, Japan).

Derivative Assay:

Article Title: The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP
Article Snippet: All GST fusions proteins were derived from CBP. .. GST-N-HAT (1096–1514 amino acids), GST-C-HAT (1514–1679 amino acids), GST-ΔC-HAT (1514–1723 amino acids) and GST-TAZ2 (1758–1894 amino acids) were constructed by PCR-amplification of pRc/RSV-CBP ( ) and cloning products into pGEX-2T (GE Healthcare) at the BamHI/EcoRI sites.

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: 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).

Transfection:

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Paragraph title: Cell culture, transfection, and siRNA ... The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Chromatography:

Article Title:
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.

Cell Culture:

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Paragraph title: Cell culture, transfection, and siRNA ... The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Article Title: β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms
Article Snippet: E. coli strains were cultured in LB-Miller medium or agar plate (BD Difco, Franklin Lakes, NJ) with appropriate antibiotics. .. For gene cloning, pGEM-T TA cloning vector (Promega, Madison, WI) was employed and pGEX-2T (GE Healthcare Life Science, Pittsburgh, PA) was used for recombinant protein expression.

Hemagglutination Assay:

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Human TSHα promoter from −802 to +22 in pGL3, rat mGPDH promoter B from −316 to +109 in pGL2, the 5xUAS-Luc reporter, and the expression plasmids of chicken TRα1 and human TRβ1 [both N-terminally fused with hemagglutinin (HA) and Flag epitopes] in pSG5 and pcDNA3 have been described previously [ , ]. .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany).

Generated:

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Various Gal4-DBD-TR fusion proteins have been designed by PCR amplification of chicken TRα fragments which were cloned into pcDNA3 (Life Technologies, Darmstadt, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany). .. Point mutations were introduced using a self-made site-directed mutagenesis system adapted from the QuickChange kit by Stratagene.

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: DNA encoding human Dlg1 PDZ2, residues 310–407, was generated by PCR sewing using oligonucleotide templates with optimal E. coli codon usage. .. The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct.

DNA Sequencing:

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany). .. Point mutations were introduced using a self-made site-directed mutagenesis system adapted from the QuickChange kit by Stratagene.

Article Title: Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins
Article Snippet: These amplicons were then digested with the indicated restriction enzymes ( ) and cloned into a tailored version of pGEX-2T (GE Healthcare) [ ]. .. These amplicons were then digested with the indicated restriction enzymes ( ) and cloned into a tailored version of pGEX-2T (GE Healthcare) [ ].

Protein Concentration:

Article Title:
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 ( , ).

Sequencing:

Article Title: The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
Article Snippet: Antisera against Francisella Lon, ClpP, ClpX, and FTL1017 were prepared with recombinant forms of these proteins in New Zealand White rabbits essentially as described previously ( ). .. Lon, ClpP, and ClpX were expressed as glutathione S -transferase (GST) fusion proteins by amplifying their coding regions from LVS genomic DNA with primer pairs Pr3230/Pr3231 ( lon ), Pr5476/Pr5477 ( clpP ), and Pr5478/Pr5479 ( clpX ) and cloning each of the PCR products in the NotI/AscI site of pST2700, a modified pGEX-2T with additional cloning sites ( ). pST2700 was constructed by insertion of an MCS sequence into the BamHI/EcoRI site of pGEX-2T. .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. GST-NBL1 protein was purified as described previously [ , ].

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence. .. GST-NBL1 protein was purified as described previously [ , ].

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Cdk8 siRNA (Cell Signaling Technology, Danvers, MA) was introduced into cells using siPORT transfection reagent (Invitrogen) according to the manufacturer's instructions. .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA). .. The fusion proteins, as well as the GST control, were expressed and purified from E. coli as suggested by the manufacturer.

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Sonication:

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. BL21 DE3 pLysS E. coli were transformed with pGEX-2T-Dlg1 PDZ2, grown in LB media under 50 µg/L ampicillin selection at 37°C to an optical density at 600 nm of 1.0 at which point protein expression was induced by the addition of 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (final concentration), the temperature was lowered to 20°C, and induction proceeded for 16 hours.

Injection:

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 purified protein was digested with thrombin (Amersham Biosciences) to remove the GST tag and re-purified using Ni-CAM™ affinity resin (Sigma).

Recombinant:

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 SCP2R primer was designed to incorporate a His6 (hexahistidine) tag into the C-terminus of the recombinant protein. .. The product was cut out with Bam HI and Eco RI from the plasmid and transferred into pGEX-2T (Amersham Biosciences).

Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: Both Pf tRip and Pf tRip1–174 were cloned into pQE30 (Qiagen) to produce proteins with a 6-histidine fusion tags at their N termini. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein. .. Thrombin cleavage occurred at Proline 214 (instead of the cleavage site provided in pGEX-2T); thus, the C-terminal domain of Pf tRip was consequently referred as Pf tRip214–402 .

Article Title: β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms
Article Snippet: E. coli strains were cultured in LB-Miller medium or agar plate (BD Difco, Franklin Lakes, NJ) with appropriate antibiotics. .. For gene cloning, pGEM-T TA cloning vector (Promega, Madison, WI) was employed and pGEX-2T (GE Healthcare Life Science, Pittsburgh, PA) was used for recombinant protein expression. .. Fold change in gene expression in cobiofilms versus single species was determined by reverse transcription-quantitative PCR (qRT-PCR) using the double delta cycle threshold (ΔΔ CT ) method as described previously ( ).

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ).

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).

Fluorescence:

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Positive transfectants were obtained by fluorescence-activated cell sorting (FACS) to generate a CCNC−/− MEF pool. .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Isolation:

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Paragraph title: Isolation and Characterization of DNA Sequences ... Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany).

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA). .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Flow Cytometry:

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. 0.2 mM phenylmethylsulfonyl fluoride (final concentration) was added during sonication and the lysate clarified by centrifugation at 23,000×g for 45 min.

Purification:

Article Title: Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly
Article Snippet: Paragraph title: Purification of Glutathione S -Transferase (GST)-recombinant Proteins ... The cDNAs encoding different portions of CPAP were fused in-frame to GST in pGEX-2T (Amersham Biosciences, Piscataway, NJ).

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: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein.

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: Paragraph title: Cloning and Purification ... The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct.

Polymerase Chain Reaction:

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: UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration
Article Snippet: A portion of cDNA encoding BICD-1(227–737) was amplified by PCR with the appropriate restriction sites and cloned into the Bam HI site of pGEX-2T to create a GST-BICD-1 fusion protein (pSL294). .. Full-length nud-2 cDNA was amplified by PCR with the appropriate overhanging restriction sites and cloned into the Pvu I and Sac I sites of pGEX-2T to create a GST-NUD-2 fusion protein (pSL246). cDNA encoding NUD-2(239–293) was amplified by PCR with MfeI and BamHI overhanging restriction sites and cloned into the EcoRI and BamHI sites of pGEX-2T to create a GST-NUD-2(239–293) fusion protein (pSL424). .. Full-length egal-1 cDNA was amplified by PCR with the appropriate overhanging restriction sites and cloned into the Bam HI and Hind III sites of pMAL-c2 (New England Biolabs, Ipswich, MA) to create an MBP-EGAL-1 fusion protein (pSL396). cDNA enoding residues 1–698 of UNC-83c were used in the MBP-UNC-83 fusion construct (pDS31; ( ).

Article Title: The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host
Article Snippet: Antisera against Francisella Lon, ClpP, ClpX, and FTL1017 were prepared with recombinant forms of these proteins in New Zealand White rabbits essentially as described previously ( ). .. Lon, ClpP, and ClpX were expressed as glutathione S -transferase (GST) fusion proteins by amplifying their coding regions from LVS genomic DNA with primer pairs Pr3230/Pr3231 ( lon ), Pr5476/Pr5477 ( clpP ), and Pr5478/Pr5479 ( clpX ) and cloning each of the PCR products in the NotI/AscI site of pST2700, a modified pGEX-2T with additional cloning sites ( ). pST2700 was constructed by insertion of an MCS sequence into the BamHI/EcoRI site of pGEX-2T. .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP
Article Snippet: Escherichia coli expression plasmids for GST, GST-HAT (1096–1757 amino acids) and GST-C/H3 (1514–1894 amino acids) have been described ( ). .. GST-N-HAT (1096–1514 amino acids), GST-C-HAT (1514–1679 amino acids), GST-ΔC-HAT (1514–1723 amino acids) and GST-TAZ2 (1758–1894 amino acids) were constructed by PCR-amplification of pRc/RSV-CBP ( ) and cloning products into pGEX-2T (GE Healthcare) at the BamHI/EcoRI sites. .. GST-HBZ and GST-HBZ-Mut(LXXAA)2 , HBZ-ΔbZIP (1–122 amino acids) and HBZ-bZIP(120–206 amino acids) , pRSETA-p53 ( ) have been described. c-Jun bZIP (257–334 amino acids) was PCR-amplified from pBiFC-bJunVN173 ( ) and cloned into pRSETA at the BamHI/EcoRI sites.

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: Various Gal4-DBD-TR fusion proteins have been designed by PCR amplification of chicken TRα fragments which were cloned into pcDNA3 (Life Technologies, Darmstadt, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany). .. Point mutations were introduced using a self-made site-directed mutagenesis system adapted from the QuickChange kit by Stratagene.

Article Title: Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins
Article Snippet: PCR amplicons corresponding to TolT molecules were cloned into the pGEM-T easy vector (Promega), and used to transform DH5α cells (Invitrogen). .. These amplicons were then digested with the indicated restriction enzymes ( ) and cloned into a tailored version of pGEX-2T (GE Healthcare) [ ].

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA). .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

Article Title: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: The Pf tRip gene was amplified by PCR from a P. falciparum cDNA library (provided by H. Vial, CNRS UMR 5235, University of Montpellier 2, Montpellier, France) and sequenced. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein.

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: DNA encoding human Dlg1 PDZ2, residues 310–407, was generated by PCR sewing using oligonucleotide templates with optimal E. coli codon usage. .. The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct.

Positron Emission Tomography:

Article Title: A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms
Article Snippet: For expression of a fusion protein with glutathione S -transferase (GST), the SoCSaseLP cDNA fragment was inserted into the Bam HI/Eco RI site of pGEX-2T (GE Healthcare Japan, Tokyo, Japan). .. The resultant plasmid, pGST-CSLP, was used for production of a GST-fused SoCSaseLP (called GST-CSLP).

Affinity Column:

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. 0.2 mM phenylmethylsulfonyl fluoride (final concentration) was added during sonication and the lysate clarified by centrifugation at 23,000×g for 45 min.

FACS:

Article Title: Cyclin C mediates stress-induced mitochondrial fission and apoptosis
Article Snippet: Positive transfectants were obtained by fluorescence-activated cell sorting (FACS) to generate a CCNC−/− MEF pool. .. The GST-cyclin C fusion genes were constructed by inserting the human cyclin C cDNA (a gift from S. Reed, Scripps Research Institute, La Jolla, CA) or the yeast coding sequence into pGEX-2T (GE Healthcare, Pittsburgh, PA).

cDNA Library Assay:

Article Title:
Article Snippet: A GST-tagged barrier to autointegration factor (GST-BAF) and vaccinia-related kinase 1 (GST-VRK1) were also produced by the same split-primer PCR method using antisense primers, AODS (for the first PCR), and AODS-3 (for the second PCR) (CellFree Sciences) and a control protein, GST-tagged bacterial dihydrofolate reductase (GST-DHFR), was produced using AODA2306 and AODA2303 from the plasmid DNAs described previously ( , , ). .. 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: Apicomplexa-specific tRip facilitates import of exogenous tRNAs into malaria parasites
Article Snippet: The Pf tRip gene was amplified by PCR from a P. falciparum cDNA library (provided by H. Vial, CNRS UMR 5235, University of Montpellier 2, Montpellier, France) and sequenced. .. The recombinant Pf tRip214–402 , corresponding to the 260-amino-acid C-terminal domain, was cloned into pGEX-2T (Amersham Biosciences) to yield an N-terminal GST fusion protein.

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).

SDS Page:

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The cells were harvested by centrifugation at 4°C and washed with cold phosphate-buffered saline (PBS) before being processed for cell lysis, SDS-PAGE, and immunoblotting. .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ).

Plasmid Preparation:

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). .. The recombinant protein was expressed using the conditions recommended by the manufacturer.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: A full-length NBL1 cDNA was used to construct the expression vector for glutathione-S-transferase (GST)-tagged NBL1 protein. .. The cDNA sequence between positions 267 and 731 was inserted into Eco RI/ Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Article Title: Impaired Repressor Function in SUMOylation-Defective Thyroid Hormone Receptor Isoforms
Article Snippet: The expression plasmid of human TRβ1 is a kind gift from Peter Hofmann (Charité Berlin, Germany). .. Various GST fusion proteins have been generated by PCR amplification of TRα or TRβ fragments and cloned into pGEX-2T (GE Healthcare, Munich, Germany).

Article Title: A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms
Article Snippet: Paragraph title: Plasmid construction ... For expression of a fusion protein with glutathione S -transferase (GST), the SoCSaseLP cDNA fragment was inserted into the Bam HI/Eco RI site of pGEX-2T (GE Healthcare Japan, Tokyo, Japan).

Article Title: Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins
Article Snippet: PCR amplicons corresponding to TolT molecules were cloned into the pGEM-T easy vector (Promega), and used to transform DH5α cells (Invitrogen). .. These amplicons were then digested with the indicated restriction enzymes ( ) and cloned into a tailored version of pGEX-2T (GE Healthcare) [ ].

Article Title: β-Glucanase Activity of the Oral Bacterium Tannerella forsythia Contributes to the Growth of a Partner Species, Fusobacterium nucleatum, in Cobiofilms
Article Snippet: E. coli strains were cultured in LB-Miller medium or agar plate (BD Difco, Franklin Lakes, NJ) with appropriate antibiotics. .. For gene cloning, pGEM-T TA cloning vector (Promega, Madison, WI) was employed and pGEX-2T (GE Healthcare Life Science, Pittsburgh, PA) was used for recombinant protein expression. .. Fold change in gene expression in cobiofilms versus single species was determined by reverse transcription-quantitative PCR (qRT-PCR) using the double delta cycle threshold (ΔΔ CT ) method as described previously ( ).

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The antisera against PtvB and SpxB were prepared with recombinant forms of the proteins in New Zealand White rabbits, essentially as described previously ( ). .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Affinity Chromatography:

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ). .. The MCS fragment was generated by annealing reverse complementary oligonucleotides Pr3110 and Pr3111.

Sampling:

Article Title: Circulating autoantibodies against neuroblastoma suppressor of tumorigenicity 1 (NBL1): A potential biomarker for coronary artery disease in patients with obstructive sleep apnea
Article Snippet: Paragraph title: Blood sampling/purification and analysis ... The cDNA sequence between positions 267 and 731 was inserted into Eco RI/Xho I site of pGEX-2T (GE Healthcare Life Sciences, Pittsburgh, PA, USA), which produces the truncated NBL1 protein (from amino acid residue 25 to 178) lacking its potential signal peptide sequence.

Concentration Assay:

Article Title: Structure of the Human Discs Large 1 PDZ2- Adenomatous Polyposis Coli Cytoskeletal Polarity Complex: Insight into Peptide Engagement and PDZ Clustering
Article Snippet: The Dlg1 PDZ2 fragment was inserted into pGEX-2T (GE Healthcare) forming a thrombin-cleavable, N-terminal GST fusion construct. .. Cells were harvested by centrifugation at 2100×g for 10 minutes and resuspended in 200 mL buffer A: 25 mM Tris pH 8.0, 300 mM NaCl, 0.1% β-mercaptoethanol, and lysed by iterative rounds of sonication at 4°C followed by sample cooling.

Lysis:

Article Title: Transcriptional Repressor PtvR Regulates Phenotypic Tolerance to Vancomycin in Streptococcus pneumoniae
Article Snippet: The cells were harvested by centrifugation at 4°C and washed with cold phosphate-buffered saline (PBS) before being processed for cell lysis, SDS-PAGE, and immunoblotting. .. PtvB and SpxB were expressed as glutathione S -transferase (GST) ( ) fusion proteins in E. coli cells by amplifying their coding regions from D39 genomic DNA with the primer sets Pr2773/Pr2774 (PtvB) and Pr3129/Pr3130 (SpxB) and cloning each of the PCR products in the NotI/AscI site of plasmid pTH2700 , a modified pGEX-2T plasmid with additional cloning sites. pTH2700 was constructed by insertion of a multiple-cloning site (MCS) sequence into the BamHI/EcoRI site of pGEX-2T (GE Healthcare BioScience, Piscataway, NJ).

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  • 87
    GE Healthcare bacterial expression vector pgex 2t
    HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors <t>pGEX-2T</t> or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa
    Bacterial Expression Vector Pgex 2t, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    GE Healthcare gst syndapin i sh3 domain
    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 <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: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    96
    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: 96/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa

    Journal: Inflammation Research

    Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies

    doi: 10.1007/s00011-017-1086-7

    Figure Lengend Snippet: HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa

    Article Snippet: Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ].

    Techniques: Expressing, Construct, Recombinant, Clone Assay, Staining, Migration, Molecular Weight

    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: 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

    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: 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: 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: 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: 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: 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

    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: 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: 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