Journal: Frontiers in Immunology

Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

doi: 10.3389/fimmu.2018.02852

Figure Lengend Snippet: Mutation of S2152A within FLNa abolishes TCR-mediated T-cell adhesion, interaction with APCs and LFA-1 activation. (A) Jurkat T cells were transiently transfected with dsRed C1 vector (dsRed) or plasmids encoding wild type (WT) dsRed-tagged FLNa (FLNa WT) or dsRed-tagged S2152A or dsRed-tagged S2152E FLNa mutants (FLNa S215A and FLNa S2152E). After 24 h the expression of WT and its mutants were analyzed by anti-dsRed and FLNa immunoblotting. Detection of β-actin served as loading control. (B) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated for 30 min with CD3 antibodies. Cells were analyzed for adhesion to ICAM-1-coated 96 well plates. Bound cells were counted and calculated as percentage of input ( n = 4) (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001). (C) Cells were transfected as described in (A) and analyzed for their ability to form conjugates with DDAO-SE (red)-stained Raji B cells that were pulsed without (non) or with superantigen (SA) for 30 min at 37°C. The percentage of conjugates was defined as the number of double-positive events in the upper right quadrant ( n = 4) (mean ± SEM; *** p ≤ 0.001). (D) Jurkat T cells transfected as described in (A) were left untreated (non) or stimulated with anti-CD3 antibodies (CD3), followed by staining with the anti-LFA-1 antibody mAb24 to detect the high affinity conformation of LFA-1. mAb24 epitope expression was assessed by flow cytometry and data are normalized against LFA-1 expression detected by MEM48 ( n = 4) (mean ± SEM; *** p ≤ 0.001). (E) HEK 293T cells were transfected with either dsRed, dsRed-tagged FLNa wild type (FLAa WT) or its mutants (FLNa S2152A and FLNa S2152E). 24 h after transfection, whole cell extracts were prepared and analyzed for the expression of dsRed and dsRed-tagged FLNa forms by Western blotting using the indicated antibodies (left panel). Lysates were incubated with GST-fusion proteins bound to glutathione-sepharose beads. Precipitates were analyzed by Western blotting using the indicated antibodies (right panel). One representative experiment of 3 is shown. (mean ± SEM; * p ≤ 0.05, *** p ≤ 0.001).

Article Snippet: Protein Purification GST, GST-tagged Igl repeats 19–24 of human FLNa and GST-tagged cytoplasmic domain of CD18 (GST-CD18cyt ) were expressed in BL21 (DE3) cells and purified using glutathione-sepharose (Novagen or GE Healthcare) according to the manufacturer's instructions.

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Cytometry, Western Blot, Incubation

Journal: Molecular and Cellular Biology

Article Title: The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

doi: 10.1128/MCB.00270-13

Figure Lengend Snippet: The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

Article Snippet: Clarified lysates were incubated with 1 ml of bovine serum albumin (BSA)-blocked 50% glutathione-Sepharose resin (GE Healthcare) for 1 h at 4°C to purify GST and GST-OAR.

Techniques: Recombinant, Incubation

Journal: Antioxidants & Redox Signaling

Article Title: Glutathione Peroxidase 7 Utilizes Hydrogen Peroxide Generated by Ero1? to Promote Oxidative Protein Folding

doi: 10.1089/ars.2013.5236

Figure Lengend Snippet: Formation of covalent binary complexes by GPx7, Ero1α, and PDI. (A) Aliquots of glutathione S-transferase (GST)-GPx7 WT, GST-GPx7 DM, or GST were incubated with HeLa cell lysates and precipitated with GSH-Sepharose. The precipitated proteins and

Article Snippet: For pulldown assay, Glutathione Sepharose resins (GE Healthcare) were incubated with 10 μ M GST-GPx7 fusion proteins and HeLa lysates (0.5 mg protein/ml) for 4 h at 4°C in PBS, and washed five times with ice-cold PBS.

Techniques: Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

doi: 10.1016/j.omtn.2018.02.011

Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

Journal: PLoS Pathogens

Article Title: The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation

doi: 10.1371/journal.ppat.1001331

Figure Lengend Snippet: K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.

Article Snippet: Immunoprecipitation, GST pulldown and immunoblot assays Proteins were extracted from cells, normalized by BCA assay (Pierce) and subjected to adsorption with protein A-agarose (SCBT) plus antibody for IP, incubat ion with glutathione-sepharose agarose (Amersham) for pulldown, or directly subjected to SDS-polyacrylamide gel electrophoresis and WB, as previously described .

Techniques: Staining, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, Construct, Purification, Western Blot, Mutagenesis

Journal: Clinical and Experimental Immunology

Article Title: The GOR gene product cannot cross-react with hepatitis C virus in humans

doi: 10.1046/j.1365-2249.2001.01508.x

Figure Lengend Snippet: The recombinant GOR1–125 protein in 15% SDS-PAGE stained with CBB. GOR1–125 protein is produced by Escherichia coli as fusion protein with GST and cleaved by site-specific protease ( see Materials and Methods). GST and protease were removed using glutathione 4B sepharose. Lane 1, purified product; size 14 kD, lane 2, 29 kD purified GST as molecular size standard, lane M, molecular size marker. Numbers on the left show molecular sizes in kD.

Article Snippet: Cell lysate was purified with glutathione 4B sepharose and digested with ProScission protease (Amersham Pharmacia Biotech) at 5°C for 4 h. Remaining protease and GST fragments were absorbed by glutathione 4B sepharose.

Techniques: Recombinant, SDS Page, Staining, Produced, Purification, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases

doi: 10.1073/pnas.1708584115

Figure Lengend Snippet: RSK2 forms a complex with RhoA GTPases. ( A ) U87MG cells transfected with the indicated Flag-RSK2 and wild-type (WT) HA-Rho GTPase constructs and serum starved. Lysates were subjected to anti–Flag-RSK2 immunoprecipitation (IP) and the coprecipitated HA-tagged Rho GTPases were detected by immunoblotting with biotinylated anti-HA antibody. The IP efficiency of Flag-RSK2 proteins and the equal loading of HA-Rho GTPases were determined. A representative of seven independent experiments is shown. ( B ) U87MG cells expressing GST-tagged WT-Rho GTPases together with Flag-RSK2-WT or Flag-RSK2-Y707A and serum starved. GST-Rho GTPases were recovered by GST pulldown, and coprecipitated Flag-RSK2 proteins were detected by immunoblotting with anti-Flag antibody. The precipitated GST-fused protein and equal loading of the Flag-RSK2 proteins are shown. The results shown are representative of three independent experiments. ( C ) U87MG cells were transfected with Flag-RSK2-WT and the indicated GST-Rho constructs and serum starved before stimulation with EGF (100 ng/mL, 5 min) or TNFα (50 ng/mL, 15 min). Lysates were subjected to GST pulldown. Coprecipitated Flag-RSK2 proteins were detected by immunoblotting with anti-Flag monoclonal antibody, while recovered GST-Rho proteins were determined by anti-GST immunoblotting. A representative of four independent experiments is shown. ( D ) U87MG cell lysate was subjected to immunoprecipitation with anti-RhoA/B/C antibody and bound proteins were recovered by incubation with protein-G Sepharose. The presence of coprecipitated RSK1/2 was determined by immunoblotting with RSK1 or -2 antibodies. The input levels of RSK1/2 and the immunoprecipitated Rho proteins are shown. nIgG, normal rabbit control IgG. A representative of four independent experiments is shown.

Article Snippet: Detergent-soluble cell lysates were prepared using kinase lysis buffer and 1–2 mg of total cell lysates were subjected to either GST pulldown using 20 μL of glutathione-coupled Sepharose (GE Healthcare), or immunoprecipitation using 2 μg of antibody together with 20 μL of protein-G Sepharose (GE Healthcare) in a final volume of 1 mL.

Techniques: Transfection, Construct, Immunoprecipitation, Expressing, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases

doi: 10.1073/pnas.1708584115

Figure Lengend Snippet: RSK2 forms a signaling complex composed of Rho GTPases and the RhoGEF, LARG. ( A ) Active RSK2 precipitates endogenous LARG. A total of 10 mg of U87MG cell lysates with overexpressed Flag-RSK2-Y707A or Flag-RSK2-T577E was incubated with anti-Flag antibody at 4 °C overnight and bound proteins were detected by immunoblotting with indicated antibodies. The arrow indicates possible phosphorylated LARG. ( B and C ) Association of RSK2 and LARG. U87MG cells were transfected with indicated Flag- or Myc-tagged constructs and serum starved for 24 h before lysate preparation. Flag ( C )- or Myc ( D )-tagged proteins were immunoprecipitated by anti-Flag or -Myc antibodies, respectively. Bound proteins were detected by immunoblotting with anti-Flag (RSK2) or -Myc (LARG) antibodies. Results are representative of three independent experiments. ( D and E ) RSK2 interacts directly with LARG. GST-LARG proteins and MBP-His 6 -RSK2-WT were incubated with either glutathione Sepharose ( D ) or Ni-NTA His-Bind resin ( E ). Bound MBP-His 6 -RSK2-WT ( D ) or GST-LARG ( E ) was determined by immunoblotting. ( F ) RSK2 phosphorylates LARG on Ser1288 residue. U87MG cells were cotransfected with Flag-RSK2 constructs and the indicated Myc-LARG constructs. The Myc-LARG proteins were immunoprecipitated after serum starvation for 24 h. LARG phosphorylation was determined by immunoblotting anti-Myc immunoprecipitates with a phospho-AKT substrate-specific antibody. Results are representative of three independent experiments.

Article Snippet: Detergent-soluble cell lysates were prepared using kinase lysis buffer and 1–2 mg of total cell lysates were subjected to either GST pulldown using 20 μL of glutathione-coupled Sepharose (GE Healthcare), or immunoprecipitation using 2 μg of antibody together with 20 μL of protein-G Sepharose (GE Healthcare) in a final volume of 1 mL.

Techniques: Incubation, Transfection, Construct, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions

doi: 10.1093/nar/gkh1000

Figure Lengend Snippet: Tfg3 is a TAF. ( A ) The Tfg3–TBP interaction. H 6 -tagged TBP was incubated with GST or GST–Tfg3 at the indicated temperatures and then treated with GSH–Sepharose. Proteins bound to the resin were analyzed by SDS–PAGE and

Article Snippet: Each recombinant protein was expressed and purified on a column of glutathione (GSH)-Sepharose 4B (Amersham) or of Ni2+ -NTA agarose (Qiagen) column essentially as described elsewhere ( ).

Techniques: Incubation, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

doi: 10.3390/ijms15022929

Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

Journal: PLoS ONE

Article Title: Anti-Aquaporin-1 Autoantibodies in Patients with Neuromyelitis Optica Spectrum Disorders

doi: 10.1371/journal.pone.0074773

Figure Lengend Snippet: Anti-AQP1 specificity of the identified autoantibodies. (A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using 125 I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using 125 I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with 125 I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.

Article Snippet: To test whether the antibodies bound to the AQP1 moiety of the AQP1-GST fusion protein, 10 µl of sera positive for anti-AQP1 autoantibodies was preincubated for 3 h at 4°C with excess GST (1.6 µg) immobilized on Sepharose-glutathione beads (GE Healthcare), then 5 µl of the treated samples was tested in the RIPA using 125 I-streptavidin-labeled AQP1-GST.

Techniques: Binding Assay, Labeling, Expressing, Transfection, Purification

Journal: Retrovirology

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

doi: 10.1186/1742-4690-3-83

Figure Lengend Snippet: Interaction between the NF90ctv RG- domain and Rev by affinity chromatography. (A) The GST/RG- recombinant protein was expressed in E. coli , the cell extracts coupled to a glutathione-Sepharose 4B column was used in pull-down assays with HeLa cell extracts previously transfected with pRSV/Rev (lane 4) or the control lysate (lane 3). A protein band corresponding to Rev (arrowhead, lane 4) was detected when extracts from HeLa cells that expressed Rev were added to the GST/RG- protein bound column; such a band was absent from control HeLa cell extract (lane 3). Lane 1, GST/RG- purified from E. coli induced by IPTG, and lane 2, purified from non induced E. coli cells. (B) Similar assays performed with purified Rev protein from E. coli in place of HeLa cells extracts. Arrow in lane 5 indicates the position of Rev. This band was not observed in absence of Rev (lane 6).

Article Snippet: The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions.

Techniques: Affinity Chromatography, Recombinant, Transfection, Purification

Journal: Infection and Immunity

Article Title: Antiapoptotic Activity of Coxiella burnetii Effector Protein AnkG Is Controlled by p32-Dependent Trafficking

doi: 10.1128/IAI.01204-13

Figure Lengend Snippet: AnkG binds directly to the host cell protein p32 and does not alter its steady-state protein level. (A) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Eluate (E1 to E4) and bead fractions were resolved by SDS-PAGE and stained with Coomassie blue. (B) Glutathione-Sepharose columns with GST-AnkG or GST alone were incubated with His-p32. Input, eluate, and bead fractions were subjected to immunoblot analysis using anti-GST and anti-p32 antibodies. (C) Ni-NTA agarose columns with His-p32 were incubated with GST or with GST-AnkG. Eluate and input were subjected to immunoblot analysis using anti-GST and anti-His antibodies. (D) HEK293 cells were transfected with plasmids encoding GFP or GFP-tagged AnkG. Protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with antibodies directed against GFP, p32, and actin. One representative immunoblot out of at least three independent experiments is shown. (E) HeLa cells were transiently transfected with plasmids encoding GFP or GFP-tagged AnkG. The cells were treated with Mitotracker (red), followed by fixation and permeabilization. p32 was stained with a specific primary antibody and a secondary dye 405-labeled antibody (blue).

Article Snippet: Purified GST or GST-AnkG was loaded onto glutathione-Sepharose columns (GE Healthcare), and purified His-p32 was added to the columns.

Techniques: Incubation, SDS Page, Staining, Transfection, Labeling

Journal: Nature cell biology

Article Title: GAPDH Mediates Nitrosylation of Nuclear Proteins

doi: 10.1038/ncb2114

Figure Lengend Snippet: SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a GSH-agarose pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.

Article Snippet: Samples were rotated for 15 min. at room temperature, 20 μl of 50% glutathione-sepharose slurry (Amersham Biosciences) were added, and samples were rotated for one additional hour at 4°C.

Techniques: Immunoprecipitation, Lysis, In Vitro, Recombinant, Binding Assay, Mutagenesis, Transfection, Western Blot

Journal: Nature cell biology

Article Title: Positive feedback between p53 and TRF2 in telomere damage signaling and cellular senescence

doi: 10.1038/ncb2123

Figure Lengend Snippet: Siah-1 interacts with and ubiquitinates TRF2. ( a ) Siah-1 interacts with TRF2 in vitro . His 6 -TRF2 was immobilized on Ni-NTA magnetic agarose beads (lanes 3–5) and incubated with GST-Siah-1 (lane 4), GST alone (lane 5) or no additional protein (lane 3). In lane 2, GST-Siah-1 was incubated with Ni-NTA magnetic agarose beads without His 6 -tagged TRF2. After extensive washing, the beads were boiled in SDS sample buffer and the eluted proteins were analyzed by immunoblot using anti-GST and anti-TRF2 antibodies. In lanes 1, 7 and 8, His 6 -TRF2, GST-Siah-1 and GST alone were run directly as input controls. A molecular weight marker was in lane 6 (MW). ( b ) Siah-1 ubiquitinates TRF2 in vitro in a RING finger-dependent manner. Rabbit reticulocyte lysates (RRL), ubiquitin, an E3 ubiquitin ligase (wild-type Siah-1, Siah-1-H59W or Siah-1-ΔRING) and a substrate (GST-TRF2 or GST) were added to the in vitro ubiquitination reaction as indicated. After reaction, glutathione-Sepharose 4FF-purified substrates were analyzed by immunoblot with anti-GST antibody. The position of non-ubiquitinated GST-TRF2 is indicated. Poly-ubiquitinated GST-TRF2 showed a smear signal (bracket) with the disappearance of non-ubiquitinated GST-TRF2. The experiment was repeated twice with reproducible results. ( c ) Siah-1 is essential to TRF2 ubiquitination in vivo . Myc-tagged TRF2, HA-tagged ubiquitin (Ub) and full-length p53 were transiently expressed in 293T cells, as indicated, which were pre-treated with control siRNA (−) or Siah-1 siRNA (#1 and #2). After treatment with MG132, protein lysates were prepared, immunoprecipitated with anti-Myc antibody or control IgG, and then analyzed by immunoblot using anti-Myc antibody (upper) and anti-HA antibody (lower). The knockdown of Siah-1 protein expression by Siah-1 siRNA was confirmed by immunoblot using total protein lysates before immunoprecipitation.β-actin was a loading control. White brackets indicate smear signals showing poly-ubiquitination. The strong signals at the bottom of the upper image correspond to IgG heavy chains. In the lower image, asterisks indicate non-specific bands. The closed arrowhead corresponded to the frontline of the electrophoresis, which likely contained non-specific signals and TRF2-associated ubiquitinated proteins of smaller size. The open arrowhead indicates a ubiquitinated protein of currently unknown origin. The experiment was repeated twice with reproducible results.

Article Snippet: In vitro protein synthesis of His6 -tagged TRF2, GST and GST-Siah-1 fusion protein was performed using Expressway cell-free E. coli expression system (Invitrogen), followed by the purification of His6 -tagged TRF2 using Ni-NTA magnetic agarose beads (Qiagen, Valencia, CA) and of GST and GST-Siah-1 using glutathione-Sepharose 4FF (GE Healthcare, Piscataway, NJ).

Techniques: In Vitro, Incubation, Molecular Weight, Marker, Purification, In Vivo, Immunoprecipitation, Expressing, Electrophoresis

Journal: Molecular Microbiology

Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

doi: 10.1111/j.1365-2958.2010.07461.x

Figure Lengend Snippet: The NPTH motif of HrcU contributes to the interaction between HrcU C and HrpB2. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. The P265G exchange abolishes the interaction between HrcU C and HrpB2. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. The P265G exchange in HrcU does not affect binding of both HpaB and HrcL to HrcU. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with E. coli lysates containing HpaB-c-Myc and HrcL-c-Myc respectively. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. D. GST–HrcU P265G does not interact with HrpB2. GST, GST–HrcU and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HrpB2-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

Techniques: Incubation, Binding Assay

Journal: Molecular Microbiology

Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

doi: 10.1111/j.1365-2958.2010.07461.x

Figure Lengend Snippet: The Y318D mutation abolishes the interaction between the C-terminal region of HrcU and both HrpB2 and HpaC. A. GST–HrcU 255–357/Y318D does not interact with HrpB2, HpaC and T3S substrates. GST, GST–HrcU 255–357 and GST–HrcU 255–357/Y318D were immobilized on glutathione sepharose and incubated with E. coli lysates containing HrpB2-c-Myc, HpaC-c-Myc, XopC-c-Myc and HpaA-c-Myc respectively. Total-cell extracts (TE) and eluted proteins (eluates) were analysed by immunoblotting, using c-Myc- and GST-specific antibodies. Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products. One representative blot probed with the GST-specific antibody is shown. B. HrcU Y318D does not interact with the putative translocon protein XopA. GST, GST–HrcU and GST–HrcU Y318D were immobilized on glutathione sepharose and incubated with XopA-c-Myc. TE and eluates were analysed as described in (A). Asterisks mark GST and GST fusion proteins; lower bands correspond to degradation products.

Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

Techniques: Mutagenesis, Incubation

Journal: Molecular Microbiology

Article Title: Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU

doi: 10.1111/j.1365-2958.2010.07461.x

Figure Lengend Snippet: The NPTH motif of HrcU is required for the interaction with the T3S4 protein HpaC. A. Amino acids 265–357 of HrcU are not sufficient for the interaction with HpaC. GST, GST–HrcU 255–357 , GST–HrcU 265–357 and GST–HrcU 268–357 were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. The total-cell extract (TE) and eluted proteins (eluates) were analysed by immunoblotting using c-Myc epitope- and GST-specific antibodies respectively. GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. B. Mutations within the NPTH motif abolish the interaction between HrcU C and HpaC. GST, GST–HrcU 255–357 , GST–HrcU 255–357/N264A , GST–HrcU 255–357/P265A and GST–HrcU 255–357/P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products. N264A, P265A and P265G mutations led to significantly reduced cleavage of GST–HrcU 255–357 and thus to enhanced amounts of the full-length fusion proteins. C. HpaC-c-Myc does not bind to the full-length HrcU protein carrying mutations within the NPTH motif. GST, GST–HrcU 255–357 , GST–HrcU N264A , GST–HrcU P265A and GST–HrcU P265G were immobilized on glutathione sepharose and incubated with an E. coli lysate containing HpaC-c-Myc. TE and eluates were analysed as described in (A). GST and GST fusion proteins are marked by asterisks; lower bands correspond to degradation products.

Article Snippet: Insoluble cell debris was removed by centrifugation and soluble GST and GST fusion proteins were immobilized on a glutathione sepharose matrix according to the manufacturer's instructions (GE Healthcare).

Techniques: Incubation

Journal: PLoS Genetics

Article Title: Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans

doi: 10.1371/journal.pgen.1003193

Figure Lengend Snippet: LlmF interacts with VeA in the yeast-two-hybrid, in vitro GST pull-down, and in vivo co-purification. (A) A directed yeast-two-hybrid approach measured protein-protein interactions and indicated LlmF interacts with VeA, but not the truncated VeA1. Yeast cells harboring the indicated bait and prey plasmids were grown in liquid shaking culture to a density of approximately 2×10 7 cells/ml and 10 µl was spotted on synthetic dropout media (SD) containing the appropriate supplements (uracil (U), tryptophan (T), leucine (L), and/or X-gal). A positive interaction results in the activation of the lacZ reporter, which turns the media blue in the presence of X-gal. (B) Recombinant GST, GST-LlmF, GST-LaeA, and GST-VelB were incubated with recombinant His 6 -VeA-S-tag and subsequently purified by glutathione sepharose 6B to look for co-purification of VeA with any of the GST labeled proteins. An immunoblot using anti-S-tag antibody detected the presence of the His 6 -VeA-S-tag protein and Ponceau stain of the membrane served as an indication of the amount of GST fusion proteins in each lane. GST tagged LlmF, VelB, and LaeA were capable of pulling down His 6 -VeA-S-tag, while GST alone did not. (C) Crude protein extracts were prepared from one-liter liquid shaking culture of each strain and subjected to the TAP protein purification protocol. The resulting eluate was electrophoresed on a 10% Bis-Tris SDS-PAGE gel and transferred to a nitrocellulose membrane where an anti-calmodulin antibody confirmed TAP-LlmF and an anti-S-tag antibody was used to detect VeA-S-tag and VeA1-S-tag. Strains used are: WT = RJMP103.5, OE-TAP- llmF veA -S-tag = RJMP249.1, and OE-TAP- llmF veA1 -S-tag = RJMP250.2.

Article Snippet: A C terminal GST tag from pJMP89 was inserted into the XhoI site of pJMP126 to construct pJMP134 (His6 -VeA-GST-S-tag), which was subsequently tandem purified with Ni-NTA resin followed by glutathione sepharose 6B (GE healthcare).

Techniques: In Vitro, In Vivo, Copurification, Activation Assay, Recombinant, Incubation, Purification, Labeling, Staining, Protein Purification, SDS Page