Structured Review

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

1) Product Images from "Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies"

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

Journal: Inflammation Research

doi: 10.1007/s00011-016-0987-1

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
Figure Legend 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

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

2) Product Images from "Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies"

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

Journal: Inflammation Research

doi: 10.1007/s00011-017-1086-7

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
Figure Legend 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

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

3) Product Images from "TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro"

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro

Journal: Molecules and Cells

doi: 10.1007/s10059-012-0232-x

(A) Schematic representation of various truncated p53 constructs and TIP30. p53 constructs were fused in frame to the 3′ end of the GST gene in the pGEX-2T vector. His-tagged constructs of TIP30 and p53 DBD or CTD were fused in frame at the 3′
Figure Legend Snippet: (A) Schematic representation of various truncated p53 constructs and TIP30. p53 constructs were fused in frame to the 3′ end of the GST gene in the pGEX-2T vector. His-tagged constructs of TIP30 and p53 DBD or CTD were fused in frame at the 3′

Techniques Used: Construct, Plasmid Preparation

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

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.561662

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

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

5) Product Images from "Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis"

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis

Journal: Protein expression and purification

doi: 10.1016/j.pep.2011.05.012

Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had
Figure Legend Snippet: Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had

Techniques Used: Expressing, Plasmid Preparation, Sequencing

Related Articles

Clone Assay:

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

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

Article Title: E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC?
Article Snippet: .. The cDNAs encoding CHIP and indicated fragments of CHIP were cloned into pGEX-2T vector (GE Healthcare). .. The expression and purification of GST fusion proteins and the GST pull-down assays were performed as previously described ( ).

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: .. CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Mutations were introduced using a two-stage PCR procedure based on the QuikChange (Stratagene, Santa Clara) site-directed mutagenesis protocol .

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: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: .. The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany). .. For expression of all GST-tagged proteins, overnight bacterial cultures were diluted 1:20 into LB medium containing 100 μg/ml ampicillin and grown at 37 °C to an A 600 of 0.6–0.8.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

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

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: .. The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). .. GST and GST-KIX fusions were expressed and purified from Escherichia coli BL21-CodonPlus-RIL (Stratagene, La Jolla, CA).

Centrifugation:

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

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

Amplification:

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

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: TIP30 and DNA sequences encoding full-length p53 (FL; residues 1–393), the p53 N-terminal domain (NTD; residues 1–93), the p53 DNA-binding domain (DBD; residues 94–312), and the p53 C-terminal domain (CTD; residues 301–393) were amplified by PCR using oligonucleotide primers, as shown in and . .. GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare).

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

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: .. The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany). .. For expression of all GST-tagged proteins, overnight bacterial cultures were diluted 1:20 into LB medium containing 100 μg/ml ampicillin and grown at 37 °C to an A 600 of 0.6–0.8.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

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

Filtration:

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

Construct:

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

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

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: .. CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Mutations were introduced using a two-stage PCR procedure based on the QuikChange (Stratagene, Santa Clara) site-directed mutagenesis protocol .

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: .. GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare). .. For SPR experiments, PCR products were digested with Nde I and Bam HI and were inserted into the corresponding sites in the pET15b vector (Novagen).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: The plasmid constructs pMK-bla and pMK-ova were kindly provided by Erwin Bohn (Institute of Medical Microbiology and Hygiene, University of Tuebingen, Tuebingen, Germany); plasmids encoding Myc-Rac1L61, Myc-Cdc42L61, and Myc-RhoAL63 were kindly provided by Dr. Pontus Aspenström (Uppsala University, Uppsala, Sweden); and GST-PAK-CRIB and GST-Rhotekin were kindly provided by Dr. John Collard (Netherlands Cancer Institute, Amsterdam, The Netherlands). .. The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany).

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: .. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer. .. GST fusion proteins were purified from cultures of E. coli DH5α cells (50 ml) expressing the receptor variants as follows.

Incubation:

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). .. Cultures were grown to an optical density of 0.6 to 0.8 at 37°C before induction with 0.5 mM IPTG and incubation at 15°C for 16 hours.

Activity Assay:

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: Paragraph title: ICP-binding activity of truncated variants of NSP4. ... These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer.

Expressing:

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis
Article Snippet: .. The expression vector pGEX-2T was purchased from GE Healthcare (Piscataway, NJ). .. Host cell BL21-CodonPlus (DE3) was purchased from Stratagene (La Jolla, CA).

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

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

Article Title: E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC?
Article Snippet: The cDNAs encoding CHIP and indicated fragments of CHIP were cloned into pGEX-2T vector (GE Healthcare). .. The expression and purification of GST fusion proteins and the GST pull-down assays were performed as previously described ( ).

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: Paragraph title: Construction of TIP30 and p53 expression plasmids ... GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare).

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: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: Paragraph title: Plasmids and Expression of Proteins ... The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany).

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat
Article Snippet: .. Restriction and modification enzymes, gluthatione sepharose and Mono-Q columns and the pGEX-2T bacterial expression vector were purchased from Amersham-Pharmacia Biotech, Uppsala, Sweden. .. Murine mAb to rat CD11b/c, OX42 (IgG2A) and the isotype-matched negative control mAb G155-178 were purchased from Pharmingen (San Jose, CA).

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: Paragraph title: Protein expression, purification, and pull-down experiments ... The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ).

Modification:

Article Title: Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat
Article Snippet: .. Restriction and modification enzymes, gluthatione sepharose and Mono-Q columns and the pGEX-2T bacterial expression vector were purchased from Amersham-Pharmacia Biotech, Uppsala, Sweden. .. Murine mAb to rat CD11b/c, OX42 (IgG2A) and the isotype-matched negative control mAb G155-178 were purchased from Pharmingen (San Jose, CA).

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: .. The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). .. GST and GST-KIX fusions were expressed and purified from Escherichia coli BL21-CodonPlus-RIL (Stratagene, La Jolla, CA).

Western Blot:

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

Transformation Assay:

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

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Crystallization Assay:

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. All experiments with human CBG were conducted with a mutant in which surface residue Lys126 was replaced by alanine in order to facilitate crystallization .

Countercurrent Chromatography:

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

Chromatography:

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

Protease Inhibitor:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

DNA Sequencing:

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: .. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer. .. GST fusion proteins were purified from cultures of E. coli DH5α cells (50 ml) expressing the receptor variants as follows.

Polymerase Chain Reaction:

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Mutations were introduced using a two-stage PCR procedure based on the QuikChange (Stratagene, Santa Clara) site-directed mutagenesis protocol .

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: .. GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare). .. For SPR experiments, PCR products were digested with Nde I and Bam HI and were inserted into the corresponding sites in the pET15b vector (Novagen).

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: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: .. The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany). .. For expression of all GST-tagged proteins, overnight bacterial cultures were diluted 1:20 into LB medium containing 100 μg/ml ampicillin and grown at 37 °C to an A 600 of 0.6–0.8.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

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

Sonication:

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). .. Harvested cells were lysed by sonication at 4°C in 200 mM NaCl, 20 mM HEPES, pH 7.5, 2 mM dithiothreitol (buffer A) supplemented with 1 mM phenylmethylsulfonyl fluoride.

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer. .. The cell suspension was sonicated (10 s, Branson sonicator, level 6) after the addition of DNase (50 μg/ml), RNase (50 μg/ml), lysozyme (100 μg/ml), and Pefabloc [4(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; Boehringer Mannheim; 0.5 mM].

Recombinant:

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

Article Title: 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: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Cellular Antioxidant Activity Assay:

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

Mutagenesis:

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: .. CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Mutations were introduced using a two-stage PCR procedure based on the QuikChange (Stratagene, Santa Clara) site-directed mutagenesis protocol .

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

Isolation:

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

Subcloning:

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

Purification:

Article Title: E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC?
Article Snippet: The cDNAs encoding CHIP and indicated fragments of CHIP were cloned into pGEX-2T vector (GE Healthcare). .. The expression and purification of GST fusion proteins and the GST pull-down assays were performed as previously described ( ).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany). .. After sonification, GST proteins were purified from bacterial lysates on glutathione-Sepharose 4B beads (GE Healthcare, Munich, Germany).

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

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: Paragraph title: Protein expression, purification, and pull-down experiments ... The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ).

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer. .. GST fusion proteins were purified from cultures of E. coli DH5α cells (50 ml) expressing the receptor variants as follows.

Sequencing:

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

cDNA Library Assay:

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

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

Chloramphenicol Acetyltransferase Assay:

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

Chromatin Immunoprecipitation:

Article Title: E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC?
Article Snippet: .. The cDNAs encoding CHIP and indicated fragments of CHIP were cloned into pGEX-2T vector (GE Healthcare). .. The expression and purification of GST fusion proteins and the GST pull-down assays were performed as previously described ( ).

SPR Assay:

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare). .. For SPR experiments, PCR products were digested with Nde I and Bam HI and were inserted into the corresponding sites in the pET15b vector (Novagen).

Plasmid Preparation:

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis
Article Snippet: .. The expression vector pGEX-2T was purchased from GE Healthcare (Piscataway, NJ). .. Host cell BL21-CodonPlus (DE3) was purchased from Stratagene (La Jolla, CA).

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

Article Title: E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKC?
Article Snippet: .. The cDNAs encoding CHIP and indicated fragments of CHIP were cloned into pGEX-2T vector (GE Healthcare). .. The expression and purification of GST fusion proteins and the GST pull-down assays were performed as previously described ( ).

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: .. CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Mutations were introduced using a two-stage PCR procedure based on the QuikChange (Stratagene, Santa Clara) site-directed mutagenesis protocol .

Article Title: TIP30 Directly Binds p53 Tumor Suppressor Protein In Vitro
Article Snippet: .. GST-tagged p53 constructs for use in GST pull-down assays were prepared by digesting PCR products with Bam HI and Eco RI and inserting into the corresponding sites in the pGEX-2T vector (GE Healthcare). .. For SPR experiments, PCR products were digested with Nde I and Bam HI and were inserted into the corresponding sites in the pET15b vector (Novagen).

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: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Cytotoxic Necrotizing Factor-Y Boosts Yersinia Effector Translocation by Activating Rac Protein *
Article Snippet: .. The gene encoding CNF-Y from Y. pseudotuberculosis YPIII (pIB1) was amplified using PCR primers 5′-CAGGATCCATGAAAAATCAATGGCAA-3′ and 5′-GACTCGAGTATCTTTTCATTTCCCCCT-3′ and cloned into the BamHI and XhoI restriction sites of vector pGEX-2T (Amersham Biosciences, Freiburg, Germany). .. For expression of all GST-tagged proteins, overnight bacterial cultures were diluted 1:20 into LB medium containing 100 μg/ml ampicillin and grown at 37 °C to an A 600 of 0.6–0.8.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

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

Article Title: Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat
Article Snippet: .. Restriction and modification enzymes, gluthatione sepharose and Mono-Q columns and the pGEX-2T bacterial expression vector were purchased from Amersham-Pharmacia Biotech, Uppsala, Sweden. .. Murine mAb to rat CD11b/c, OX42 (IgG2A) and the isotype-matched negative control mAb G155-178 were purchased from Pharmingen (San Jose, CA).

Article Title: Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice
Article Snippet: .. The cDNAs encoding wild-type and Y630N murine p300 KIX domains (residues 567-667) were cloned into a modified pGEX-2T vector (GE Healthcare, Piscataway, NJ). .. GST and GST-KIX fusions were expressed and purified from Escherichia coli BL21-CodonPlus-RIL (Stratagene, La Jolla, CA).

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: .. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer. .. GST fusion proteins were purified from cultures of E. coli DH5α cells (50 ml) expressing the receptor variants as follows.

Negative Control:

Article Title: Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat
Article Snippet: Restriction and modification enzymes, gluthatione sepharose and Mono-Q columns and the pGEX-2T bacterial expression vector were purchased from Amersham-Pharmacia Biotech, Uppsala, Sweden. .. Murine mAb to rat CD11b/c, OX42 (IgG2A) and the isotype-matched negative control mAb G155-178 were purchased from Pharmingen (San Jose, CA).

Binding Assay:

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: Previous studies of the binding of NSP4 and rotavirus ICPs revealed the importance of the extreme C terminus for receptor activity ( , ) and demonstrated that GST fusion proteins containing as little as the C-terminal 20 amino acids retain the ability to bind ICPs when immobilized on the surface of glutathione-agarose beads ( , ). .. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer.

Two Hybrid Screening:

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

Produced:

Article Title: Corticosteroid-Binding Globulin: Structure-Function Implications from Species Differences
Article Snippet: CBG plasmid constructs and site-directed mutagenesis The coding sequences of residues 11 to 383 of human CBG (Swiss-Prot entry P08185) and residues 5 to 374 of rat CBG (P31211) were cloned into a pGEX-2T vector (GE Healthcare, Uppsala), yielding N-terminal glutathione-S-transferase (GST) fusion proteins. .. Wild-type rat CBG (rat CBG-WT) and rat CBG mutants CBG-RCL1, CBG-RCL2 and CBG-RCL3 were produced to probe the function of the RCL residues in rat CBG.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments 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 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Lysis:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

Variant Assay:

Article Title: Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle
Article Snippet: A C16 variant was constructed by using the protocol described previously for the C90 to C20 variants ( ). .. These cDNA fragments were cloned into the pGEX-2T vector (Amersham Pharmacia Biotech), and the identities of the constructs were confirmed by DNA sequencing using an Applied Biosystems automated sequencer.

HMT Assay:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

    Techniques: Binding Assay, Mutagenesis, In Vitro, Purification

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

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

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

    Techniques: Patch Clamp, Transfection

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

    Techniques: Transfection, Immunolabeling, Quantitation Assay, Expressing

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

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

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

    Techniques: Binding Assay, Mutagenesis, In Vitro, Purification

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

    Journal: The Journal of Cell Biology

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

    doi: 10.1083/jcb.201307088

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

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

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

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

    Journal: Inflammation Research

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

    doi: 10.1007/s00011-016-0987-1

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

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

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

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

    Journal: The Journal of Clinical Investigation

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

    doi: 10.1172/JCI127277

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

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

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