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  • 95
    Thermo Fisher gst tag polyclonal antibody
    Validation of PVX110940 in plasma-derived exosomes from P. vivax infected FRG huHep mice. (A) Schematic representation of full-length PVX110940 protein and a recombinant <t>GST-fusion</t> truncated (GST-PVX110940-Tr) version containing a predicted B-cell antigenic region of 156 amino acids. Red square represents a predicted N-terminal signal peptide. (B) GST-PV110940-Tr was produced from two E. coli clones (C11 and C25) in a wheat germ cell-free system and affinity-purified products resolved on SDS-PAGE. Western blot analysis using monoclonal anti-GST antibodies. (C) Recombinant GST-PVX110940-Tr was used to immunize mice and produce <t>polyclonal</t> antibodies. Western blot analysis shows recognition of GST-PVX110940-Tr purified recombinant protein and purified GST. (D) Flow cytometry bead-based assay showing PVX110940 detection in ExEF from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Beads were incubated with the highest-CD5L SEC fraction of each sample and used for detection of PVX110940 using the polyclonal antibodies raised in mice. Mean fluorescence intensity (MFI) of PVX110940 and controls was assayed. Specificity controls were as follows: C1: Highest-CD5L SEC fraction of each sample incubated with Alexa-488 mouse secondary antibody. C2: Pool of CD5L highest fraction of all samples incubated with isotype rabbit antibody and Alexa-488 mouse secondary antibody. Stars indicate the samples were positive detection was observed over the background signal observed in uninfected mice (Dashed line). (E) Western blot analysis of PVX-110940- in isolated exosomes (CD5L-highest SEC fraction) derived from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Hundred micro liters of CD5L-highest fraction were blotted. Membranes were incubated with 1/50 of polyclonal anti-PVX110940 antibody. * Samples where a positive signal at the expected molecular size (84 kDa) is observed.
    Gst Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore gst tag
    IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and <t>GST-fused</t> truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, <t>detagged</t> TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.
    Gst Tag, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gst tagged proteins
    <t>NHERF-2</t> interacts with β-catenin in vitro and in vivo. (A) Schematic of tagged NHERF-2 proteins, indicating the tag, the two PDZ domains, and the ERM-binding domain (ERM BD). (B) An equal amount of each <t>GST-tagged</t> NHERF protein was immobilized
    Gst Tagged Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 873 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore gst proteins
    <t>CUGBP2</t> binds directly to SF3b145 and SF3b49. ( A ) <t>GST-CUGBP2</t> and GST-CUGBP1 (Coomassie staining gels left side or each panel) coupled on glutathione-sepharose were incubated with [ 35 S] Methionine/Cysteine-labeled SF3b145 (A) and SF3b49 ( B ). Bound proteins were separated on 10% SDS–PAGE gel and visualized with Cyclone Phosphorimager. ( C ) RNase A (50 μg/ml) reduced interactions between CELF and SF3b proteins in vitro . ( D ) Bound proteins in GST pull down were treated with the reversible protein cross-linker (DSP) prior to RNase A treatment. M: protein marker.
    Gst Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare gst tagged
    A short region immediately C-terminal to the charged region of <t>SF3a120</t> is sufficient for SF3a66 binding. A , scheme of in vitro -translated and <t>GST-tagged</t> SF3a120 proteins. Boxes indicate known protein domains as follows: S1 and S2 , SURP1 and SURP 2 motifs;
    Gst Tagged, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare gst tag
    β2-β2 strand amino acids mediate <t>A3H</t> dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of <t>GST-A3H</t> haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    Gst Tag, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 6709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gst tag
    ARF6 is regulated differently in CNS vs . PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of fluoresent signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (+DIV10) labelled with <t>GGA3-ABD-GST</t> to detect active <t>ARF.</t> Graph shows quantification of ARF activation in the two axon types. n=58(DRG) and 60(cortical). ***p
    Gst Tag, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore glutathione s transferase gst tag
    ARF6 is regulated differently in CNS vs . PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of fluoresent signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (+DIV10) labelled with <t>GGA3-ABD-GST</t> to detect active <t>ARF.</t> Graph shows quantification of ARF activation in the two axon types. n=58(DRG) and 60(cortical). ***p
    Glutathione S Transferase Gst Tag, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher anti gst
    LegK2 interacts with ARPC1B and ARP3 subunits of the ARP2/3 complex. (A) Yeast two-hybrid assays of L. pneumophila protein kinase LegK1/LegK2 and ARPC1B/ARP3 subunits of the human ARP2/3 complex. Diploids from the mating of AH109(pGBKT7-legK1) or AH109(pGBKT7-legK2) with Y187(pACT2), Y187(pACT2-ARPC1B), or Y187(pACT2-ACTR3) were grown on SD medium without tryptophan (−W) or leucine (−L). Histidine auxotrophy was tested by plating 5 or 50 µl of diploids on SD-W-L medium without histidine (−H) in the presence of 10 mM 3-AT. (B) Affinity copurification of Flag-tagged ARP2/3 subunits with <t>GST-tagged</t> LegK2 protein. HEK293T cells were cotransfected with pDEST27, pDEST27-legK2, or pDEST27-legK2 K112M and pCI-Neo3Flag-ARPC1B or -ACTR3. GST, GST-tagged LegK2, or LegK2 K112M was purified on glutathione-agarose 4B, and purified fractions were <t>immunoblotted</t> with both anti-GST and anti-Flag antibodies. (C) Cellular localization of ARPC1B/ARP3 and LegK2/LegK1 proteins in HEK293T cells cotransfected with pDEST27, pDEST27-legK2, or -legK1 and pCI-Neo3Flag-ARPC1B or -ACTR3. The GST, GST-LegK2, and GST-LegK1 proteins were detected by immunofluorescence with anti-GST antibodies (green), and 3Flag-ARPC1B and 3Flag-ARP3 were detected with anti-Flag antibodies (red). Scale bars, 10 µm. (D) Quantitation of colocalization by Pearson coefficient. The Pearson coefficient was determined with the JACoP plugin of the ImageJ software and is expressed as the mean value calculated for 30 cells.
    Anti Gst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology gst tag
    <t>Mad2</t> is a new substrate for CCP6 in MKs. (A) Protein polyglutamylation was assessed by immunoblotting with GT335 antibody. Spleen and BM lysates from CCP6 -deficient and littermate control mice were analyzed. Data were repeated three times with similar results. (B) Recombinant CCP6-wt and inactive CCP6 mutant (CCP6-mut) were immobilized with Affi-gel10 resin, to which mouse BM lysates were added for affinity chromatography. The eluted fractions were visualized by SDS-PAGE, followed by silver staining. M: molecular weight marker. The differential band of 30 kD seen in CCP6-mut gel was cut for mass spectrometry and identified as Mad2. The peptide sequences and coverage analyzed by LC-LTQ MS/MS are shown in the bottom graph. (C) Glutamylated rGST-Mad2 binding to CCP6-mut protein was analyzed by <t>GST</t> pulldown. rGST-Mad2 protein was incubated with lysates from Myc-tagged CCP6-wt or Myc-tagged CCP6-mut expressed 293T cells at 37°C for 2 h, followed by incubation with GST beads. Ratios of pulldown/input of CCP6 were calculated and shown as means ± SD. **, P
    Gst Tag, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher glutathione s transferase gst tag
    <t>Mad2</t> is a new substrate for CCP6 in MKs. (A) Protein polyglutamylation was assessed by immunoblotting with GT335 antibody. Spleen and BM lysates from CCP6 -deficient and littermate control mice were analyzed. Data were repeated three times with similar results. (B) Recombinant CCP6-wt and inactive CCP6 mutant (CCP6-mut) were immobilized with Affi-gel10 resin, to which mouse BM lysates were added for affinity chromatography. The eluted fractions were visualized by SDS-PAGE, followed by silver staining. M: molecular weight marker. The differential band of 30 kD seen in CCP6-mut gel was cut for mass spectrometry and identified as Mad2. The peptide sequences and coverage analyzed by LC-LTQ MS/MS are shown in the bottom graph. (C) Glutamylated rGST-Mad2 binding to CCP6-mut protein was analyzed by <t>GST</t> pulldown. rGST-Mad2 protein was incubated with lysates from Myc-tagged CCP6-wt or Myc-tagged CCP6-mut expressed 293T cells at 37°C for 2 h, followed by incubation with GST beads. Ratios of pulldown/input of CCP6 were calculated and shown as means ± SD. **, P
    Glutathione S Transferase Gst Tag, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore gst tagged
    Densitometric analysis of westerns blots of total <t>p38</t> MAPK expression in hearts from wild-type ( WT ) and knockout (KO) mice, immunoblotted against known quantities of <t>GST-tagged</t> recombinant p38 MAPK ( n = 6/recombinant protein or heart sample). Error bars represent SEM. A representative western blot is shown in the lower panel
    Gst Tagged, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore human mdm2
    A schematic model for the function of the <t>L11-MDM2-p53</t> pathway. (See text for discussion.)
    Human Mdm2, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc gst tag
    Inhibitory effects of NTZ on the <t>HBx–DDB1</t> interaction in vitro. <t>GST-tagged</t> recombinant DDB1 protein and untagged recombinant HBx protein were mixed in vitro. NTZ or DMSO was added to the mixture and incubated for 20 minutes, followed by pull-down using anti-GST antibody and Western blotting to determine the levels of DDB1 and HBx in the pulled-down samples. Representative results of 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; * P = 7.1 × 10 –5 ( t test).
    Gst Tag, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 139 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abnova gst tag
    <t>Annexin</t> A2 expression in ovarian cancer cell lines, peritoneal cell line and co-cultured ovarian cancer and peritoneal cells (A) Annexin A2 expression in OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9 cell lines determined by real-time PCR and were assessed using 2 −ΔΔCT quantitation method. Data represents triplicate determinations ± SEM from 2 independent experiments. (B) Western immunoblotting shows annexin A2 band at 37 kDa in cell lysates of OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9. β-actin was used as loading control. (C) Western immunoblotting shows two isoforms of annexin A2 at 37 kDa and 36 kDa bands in the conditioned media (CM) of LP-9 cells alone and a 35 kDa annexin A2 band present in the CM of co-cultured LP-9 and ovarian cancer cells. (D) 2D-western immunoblotting showed multiple annexin A2 spots at 37 kDa in the CM of LP-9 cells alone and annexin A2 isoforms at 37 kDa and 35 kDa in the CM of co-cultured LP-9 and OVCAR-5 cells over a pI values range of 6 to 8. (E) Annexin A2 western immunoblotting of recombinant annexin A2 with <t>GST</t> tag (~63.4 kDa) and a cleaved annexin A2 at ~36 kDa in the presence of plasmin that was partially inhibited by α2-antiplasmin. (F) Annexin A2 western immunoblotting of the CM of co-cultured OVCAR-5 and LP-9 cells treated with α2-antiplasmin, protease inhibitor cocktail, ε-aminocaproic acid (ε-ACA), GM-6001 and DMSO.
    Gst Tag, supplied by Abnova, used in various techniques. Bioz Stars score: 93/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher gst
    <t>G3BP1</t> and G3BP2 interact with thousands of transcripts. ( a ) The G3BP1 domain structure. ( b ) <t>GST</t> western blot showing RNA-sequence-context-dependent inhibition of GST-G3BP1 binding to RNA by m 6 A. Molecular weights are indicated on the right. ( c , d ) RNA cross-link immunoprecipitation in HEK293 cells expressing either Flag-G3BP1 or Flag-G3BP2. Flag western blots ( c ) and radioactively labeled RNA-protein complexes ( d ) are shown. OE, overexpression; Ctrl, control. ( e ) Common peaks between PAR-CLIP biological replicates 1 and 2 for G3BP1 (top left) and G3BP2 (top right), and comparisons between G3BP1 and G3BP2 peaks (bottom left) and target genes (bottom right). ( f ) Metagene profiles of G3BP1 and G3BP2 distribution across the transcriptome. ( g ) The most enriched consensus sequences of G3BP1 (top) and G3BP2 (bottom) on mRNA; P values were determined as described in the Online Methods. ( h ) The most significantly enriched GO terms among G3BP1 target genes. The numbers to the right of the bars represent the number of genes in that GO term. ( i ) The number of G3BP1 and G3BP2 peaks that overlap with m 6 A peaks among m 6 A-containing mRNAs. ( j ) The number of G3BP1 and G3BP2 peaks on m 6 A-containing mRNAs that overlap with m 6 A. ( k ) The distribution of G3BP1 (left) and G3BP2 (right) relative to m 6 . The distance between PAR-CLIP peaks and m 6 A sites was counted in 10-nucleotide bins. A Gaussian kernel smoothing over the histogram is plotted as a transparent black line. The uncropped blot image for b . Source data for i and j .
    Gst, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore anti gst tag
    ABI1 inhibits <t>MKKK18</t> activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf <t>GST–ABI1</t> or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The 32 P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t -test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro . GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [ 32 P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.
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    94
    Proteintech rabbit gst tag polyclonal
    ABI1 inhibits <t>MKKK18</t> activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf <t>GST–ABI1</t> or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The 32 P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t -test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro . GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [ 32 P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.
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    Image Search Results


    Validation of PVX110940 in plasma-derived exosomes from P. vivax infected FRG huHep mice. (A) Schematic representation of full-length PVX110940 protein and a recombinant GST-fusion truncated (GST-PVX110940-Tr) version containing a predicted B-cell antigenic region of 156 amino acids. Red square represents a predicted N-terminal signal peptide. (B) GST-PV110940-Tr was produced from two E. coli clones (C11 and C25) in a wheat germ cell-free system and affinity-purified products resolved on SDS-PAGE. Western blot analysis using monoclonal anti-GST antibodies. (C) Recombinant GST-PVX110940-Tr was used to immunize mice and produce polyclonal antibodies. Western blot analysis shows recognition of GST-PVX110940-Tr purified recombinant protein and purified GST. (D) Flow cytometry bead-based assay showing PVX110940 detection in ExEF from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Beads were incubated with the highest-CD5L SEC fraction of each sample and used for detection of PVX110940 using the polyclonal antibodies raised in mice. Mean fluorescence intensity (MFI) of PVX110940 and controls was assayed. Specificity controls were as follows: C1: Highest-CD5L SEC fraction of each sample incubated with Alexa-488 mouse secondary antibody. C2: Pool of CD5L highest fraction of all samples incubated with isotype rabbit antibody and Alexa-488 mouse secondary antibody. Stars indicate the samples were positive detection was observed over the background signal observed in uninfected mice (Dashed line). (E) Western blot analysis of PVX-110940- in isolated exosomes (CD5L-highest SEC fraction) derived from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Hundred micro liters of CD5L-highest fraction were blotted. Membranes were incubated with 1/50 of polyclonal anti-PVX110940 antibody. * Samples where a positive signal at the expected molecular size (84 kDa) is observed.

    Journal: Frontiers in Microbiology

    Article Title: Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice

    doi: 10.3389/fmicb.2018.01271

    Figure Lengend Snippet: Validation of PVX110940 in plasma-derived exosomes from P. vivax infected FRG huHep mice. (A) Schematic representation of full-length PVX110940 protein and a recombinant GST-fusion truncated (GST-PVX110940-Tr) version containing a predicted B-cell antigenic region of 156 amino acids. Red square represents a predicted N-terminal signal peptide. (B) GST-PV110940-Tr was produced from two E. coli clones (C11 and C25) in a wheat germ cell-free system and affinity-purified products resolved on SDS-PAGE. Western blot analysis using monoclonal anti-GST antibodies. (C) Recombinant GST-PVX110940-Tr was used to immunize mice and produce polyclonal antibodies. Western blot analysis shows recognition of GST-PVX110940-Tr purified recombinant protein and purified GST. (D) Flow cytometry bead-based assay showing PVX110940 detection in ExEF from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Beads were incubated with the highest-CD5L SEC fraction of each sample and used for detection of PVX110940 using the polyclonal antibodies raised in mice. Mean fluorescence intensity (MFI) of PVX110940 and controls was assayed. Specificity controls were as follows: C1: Highest-CD5L SEC fraction of each sample incubated with Alexa-488 mouse secondary antibody. C2: Pool of CD5L highest fraction of all samples incubated with isotype rabbit antibody and Alexa-488 mouse secondary antibody. Stars indicate the samples were positive detection was observed over the background signal observed in uninfected mice (Dashed line). (E) Western blot analysis of PVX-110940- in isolated exosomes (CD5L-highest SEC fraction) derived from plasma of P. vivax infected FRG huHep mice from EI1 and EI2. Hundred micro liters of CD5L-highest fraction were blotted. Membranes were incubated with 1/50 of polyclonal anti-PVX110940 antibody. * Samples where a positive signal at the expected molecular size (84 kDa) is observed.

    Article Snippet: After washes, blots were incubated for 1 h with primary antibodies [rabbit anti-GST (1:5000, Invitrogen: A5800), mouse anti-GST-PVX110940-Tr (1/500 or 1/50), rabbit anti-arginase I (1/1000) (Genentex: GTX109242)] in antibodies buffer (1X PBS, 0.1% Tween-20, 1% milk powder).

    Techniques: Derivative Assay, Infection, Mouse Assay, Recombinant, Produced, Clone Assay, Affinity Purification, SDS Page, Western Blot, Purification, Flow Cytometry, Cytometry, Bead-based Assay, Incubation, Size-exclusion Chromatography, Fluorescence, Isolation

    IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and GST-fused truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, detagged TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.

    Journal: The Journal of Clinical Investigation

    Article Title: IQGAP1 suppresses T?RII-mediated myofibroblastic activation and metastatic growth in liver

    doi: 10.1172/JCI63836

    Figure Lengend Snippet: IQGAP1 C terminus aa 1503–1657 is required for binding and suppressing TβRII. ( A ) Top 4 rows: full-length (FL) IQGAP1 and GST-fused truncated IQGAP1 proteins are shown. Bottom, GST fused truncated IQGAP1 proteins extracted from bacteria were incubated with HSC lysates for GST pull-down assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII. Ponceau S staining depicted the purity of the recombinant proteins. ( B ) Top: after the GST tag of GST-TRII was removed by thrombin treatment, detagged TβRII was incubated with GST-fused IQGAP1 proteins for in vitro binding assays. Both aa 746–1657 and aa 1503–1657 of IQGAP1 bound to TβRII directly in vitro. Ponceau S staining depicted the purity of GST and GST-fused IQGAP1 proteins. Bottom: detagged IQGAP1 aa 746–1657 was incubated with GST or GST-TβRII for in vitro binding assays. GST-TβRII bound to IQGAP1 aa 746-1657 directly in vitro. aa 746–1657 instead of aa 1503–1657 of IQGAP1 was used in this assay because IQGAP1 antibodies could not recognize aa 1503–1657 of IQGAP1. Ponceau S staining depicted the purity of GST and GST fusion proteins. ( C ) HSCs expressing TβRII-HA were transduced with lentiviruses encoding GFP, IQGAP1-FLAG, or IQGAP1 (1-1502)-FLAG, and subjected to WB. In contrast with IQGAP1, IQGAP1 (1-1502) mutant lacking the TβRII binding region failed to repress TβRII protein levels. Densitometric ratios are shown on the bottom. All data shown represent multiple repeats with similar results.

    Article Snippet: For in vitro binding assays, the GST tag was first removed by thrombin, and detagged proteins were purified and recovered by the Thrombin Cleavage Capture Kit (Novagen, 69022).

    Techniques: Binding Assay, Incubation, Staining, Recombinant, In Vitro, Expressing, Transduction, Western Blot, Mutagenesis

    NHERF-2 interacts with β-catenin in vitro and in vivo. (A) Schematic of tagged NHERF-2 proteins, indicating the tag, the two PDZ domains, and the ERM-binding domain (ERM BD). (B) An equal amount of each GST-tagged NHERF protein was immobilized

    Journal:

    Article Title:

    doi: 10.1091/mbc.E06-10-0960

    Figure Lengend Snippet: NHERF-2 interacts with β-catenin in vitro and in vivo. (A) Schematic of tagged NHERF-2 proteins, indicating the tag, the two PDZ domains, and the ERM-binding domain (ERM BD). (B) An equal amount of each GST-tagged NHERF protein was immobilized

    Article Snippet: GST-tagged proteins consisting of NHERF-1 and various regions of NHERF-2, including full-length protein, the first PDZ domain, the second PDZ domain including the C terminus, both PDZ domains alone, and the C terminus alone, were purified on a glutathione column (Sigma-Aldrich), washed with Tris-buffered saline (10 mM Tris-HCl, pH 8.0, and 9 g/l NaCl) and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0.

    Techniques: In Vitro, In Vivo, Binding Assay

    Mutational analysis of RacC. ( a ) Alignment of various Rac proteins. The position of the mutations generated is indicated as well as of the Switch I and II regions. ( b ) Surface potential of D. discoideum Rac1a and RacC. The three dimensional structure of Rac1a and RacC is modeled using SWISSMODEL and the surface potential calculated using APBS plug-in in Pymol. Red, negative, blue, positive charge. ( c ) Interactions of RacC Mut1-5 with GFP-CRN7 WT. The RacC mutant proteins were expressed as GST fusions, bound to Glutathione-Sepharose beads, loaded with GDP or GTPγS and used to pull down GFP-CRN7 WT from cell lysates. C, represents the protein amounts in 2 × 10 5 cells; I, aliquot of the cell lysate used in the assay. The western blots were probed with mAb K3-184-2. ( d ) Binding affinities of different Rac mutants to GFP-CRN7. Bar chart shows the binding of RacC wild type and mutant proteins loaded with GDP or GTPγS to GFP-CRN7. Between three and five experiments were carried out. Relative intensities are shown as arbitrary units ± SD. The differences for wild type RacC (WT), Mut2 and Mut3 were statistically significant (P

    Journal: Scientific Reports

    Article Title: Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins

    doi: 10.1038/srep14437

    Figure Lengend Snippet: Mutational analysis of RacC. ( a ) Alignment of various Rac proteins. The position of the mutations generated is indicated as well as of the Switch I and II regions. ( b ) Surface potential of D. discoideum Rac1a and RacC. The three dimensional structure of Rac1a and RacC is modeled using SWISSMODEL and the surface potential calculated using APBS plug-in in Pymol. Red, negative, blue, positive charge. ( c ) Interactions of RacC Mut1-5 with GFP-CRN7 WT. The RacC mutant proteins were expressed as GST fusions, bound to Glutathione-Sepharose beads, loaded with GDP or GTPγS and used to pull down GFP-CRN7 WT from cell lysates. C, represents the protein amounts in 2 × 10 5 cells; I, aliquot of the cell lysate used in the assay. The western blots were probed with mAb K3-184-2. ( d ) Binding affinities of different Rac mutants to GFP-CRN7. Bar chart shows the binding of RacC wild type and mutant proteins loaded with GDP or GTPγS to GFP-CRN7. Between three and five experiments were carried out. Relative intensities are shown as arbitrary units ± SD. The differences for wild type RacC (WT), Mut2 and Mut3 were statistically significant (P

    Article Snippet: Pull downs were performed with Glutathione Sepharose 4B beads (GE Healthcare) carrying GST tagged fusion proteins, immunoprecipitations were done with Protein A-Sepharose 4B beads (Sigma) carrying appropriate antibodies.

    Techniques: Generated, Mutagenesis, Western Blot, Binding Assay

    The CRIB domains of CRN7 and Rac GTPase interactions of CRN7. (aa) Domain structure of coronin and CRN7. (ab) Sequence alignment of the CRIB domain in coronin and CRN7 and selected proteins. The consensus sequence is shown above the alignment. The position within the proteins is indicated. (ac) Model of CRN7. The 3D structure was predicted as described 11 . The CRIB domains are in shown red. A magnification of the region containing the CRIB domain and the putative F-actin binding site in WD2 is shown below. The conserved K residue is indicated in a stick model in magenta. ( b ) CRN7 interacts primarily with GDP-bound Rac. GST and GST-Rac proteins loaded with GDP or GTPγS were bound to Glutathione Sepharose beads and incubated with lysates of corB − cells expressing GFP-CRN7 WT. The bound proteins were analyzed by western blotting. Probing was with GFP specific mAb K3-184-2. ( c ) The N- and the C-terminal half of CRN7 interact with Rac GTPases. The precipitated proteins were detected with mAb K3-184-2, the GST-fusions with polyclonal GST-specific antibodies. GST control (GST CTL) and beads control (Beads CTL) are shown. ( d ) CRN7 interacts directly with Rac proteins. Bacterially expressed GST-CRN7-NT encompassing the PST-domain was cleaved from the GST part and used in pull down assays with GST-Rac GTPases. The precipitated proteins were analyzed in western blots. CRN7-NT was detected with mAb K67-31-5.

    Journal: Scientific Reports

    Article Title: Coronin7 regulates WASP and SCAR through CRIB mediated interaction with Rac proteins

    doi: 10.1038/srep14437

    Figure Lengend Snippet: The CRIB domains of CRN7 and Rac GTPase interactions of CRN7. (aa) Domain structure of coronin and CRN7. (ab) Sequence alignment of the CRIB domain in coronin and CRN7 and selected proteins. The consensus sequence is shown above the alignment. The position within the proteins is indicated. (ac) Model of CRN7. The 3D structure was predicted as described 11 . The CRIB domains are in shown red. A magnification of the region containing the CRIB domain and the putative F-actin binding site in WD2 is shown below. The conserved K residue is indicated in a stick model in magenta. ( b ) CRN7 interacts primarily with GDP-bound Rac. GST and GST-Rac proteins loaded with GDP or GTPγS were bound to Glutathione Sepharose beads and incubated with lysates of corB − cells expressing GFP-CRN7 WT. The bound proteins were analyzed by western blotting. Probing was with GFP specific mAb K3-184-2. ( c ) The N- and the C-terminal half of CRN7 interact with Rac GTPases. The precipitated proteins were detected with mAb K3-184-2, the GST-fusions with polyclonal GST-specific antibodies. GST control (GST CTL) and beads control (Beads CTL) are shown. ( d ) CRN7 interacts directly with Rac proteins. Bacterially expressed GST-CRN7-NT encompassing the PST-domain was cleaved from the GST part and used in pull down assays with GST-Rac GTPases. The precipitated proteins were analyzed in western blots. CRN7-NT was detected with mAb K67-31-5.

    Article Snippet: Pull downs were performed with Glutathione Sepharose 4B beads (GE Healthcare) carrying GST tagged fusion proteins, immunoprecipitations were done with Protein A-Sepharose 4B beads (Sigma) carrying appropriate antibodies.

    Techniques: Sequencing, Binding Assay, Incubation, Expressing, Western Blot, CTL Assay

    CUGBP2 binds directly to SF3b145 and SF3b49. ( A ) GST-CUGBP2 and GST-CUGBP1 (Coomassie staining gels left side or each panel) coupled on glutathione-sepharose were incubated with [ 35 S] Methionine/Cysteine-labeled SF3b145 (A) and SF3b49 ( B ). Bound proteins were separated on 10% SDS–PAGE gel and visualized with Cyclone Phosphorimager. ( C ) RNase A (50 μg/ml) reduced interactions between CELF and SF3b proteins in vitro . ( D ) Bound proteins in GST pull down were treated with the reversible protein cross-linker (DSP) prior to RNase A treatment. M: protein marker.

    Journal: Nucleic Acids Research

    Article Title: CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA

    doi: 10.1093/nar/gkp346

    Figure Lengend Snippet: CUGBP2 binds directly to SF3b145 and SF3b49. ( A ) GST-CUGBP2 and GST-CUGBP1 (Coomassie staining gels left side or each panel) coupled on glutathione-sepharose were incubated with [ 35 S] Methionine/Cysteine-labeled SF3b145 (A) and SF3b49 ( B ). Bound proteins were separated on 10% SDS–PAGE gel and visualized with Cyclone Phosphorimager. ( C ) RNase A (50 μg/ml) reduced interactions between CELF and SF3b proteins in vitro . ( D ) Bound proteins in GST pull down were treated with the reversible protein cross-linker (DSP) prior to RNase A treatment. M: protein marker.

    Article Snippet: Supernatants were collected after centrifugation at 14 800× g for 10 min. His-tagged CUGBP2 was induced like a GST proteins and purified using the Novagen kit according to the manufacture's protocol.

    Techniques: Staining, Incubation, Labeling, SDS Page, In Vitro, Marker

    Antigen-specific proliferation of CD4 + and CD8 + T cells in immunized mice . Splenocytes from PE_PGRS33-immunized mice were stained with CFSE and incubated with 25 μg of PE_PGRS33, PGRS, PE, or GST protein plus 10 μg of Polymyxin B for 4 days. Spleen cells from mice injected with only nitrocellulose were also cultured with antigens (control). Cells without the antigens were incubated for the same length of time (unstimulated). Splenocytes were then labeled with anti-CD4-phycoerythrin (A) or anti-CD8-allophycocyanin (B) monoclonal antibodies and the percentage of proliferating cells were determined by CFSE dilution and flow cytometry. Each bar represents the mean ± SD of data from four mice per group, and the results are representative of those obtained from three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    doi: 10.3389/fimmu.2014.00236

    Figure Lengend Snippet: Antigen-specific proliferation of CD4 + and CD8 + T cells in immunized mice . Splenocytes from PE_PGRS33-immunized mice were stained with CFSE and incubated with 25 μg of PE_PGRS33, PGRS, PE, or GST protein plus 10 μg of Polymyxin B for 4 days. Spleen cells from mice injected with only nitrocellulose were also cultured with antigens (control). Cells without the antigens were incubated for the same length of time (unstimulated). Splenocytes were then labeled with anti-CD4-phycoerythrin (A) or anti-CD8-allophycocyanin (B) monoclonal antibodies and the percentage of proliferating cells were determined by CFSE dilution and flow cytometry. Each bar represents the mean ± SD of data from four mice per group, and the results are representative of those obtained from three independent experiments.

    Article Snippet: Viable cells were counted by trypan blue exclusion, and 1.5 million cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B (Calbiochem, Pacific Center, CA, USA) in 24-well plates at 37°C in 5% CO2 for 96 h. Polymyxin B was added to rule out the possibility of contamination with lipopolysaccharide (LPS).

    Techniques: Mouse Assay, Staining, Incubation, Injection, Cell Culture, Labeling, Flow Cytometry, Cytometry

    Antigen-specific antibody response in LTBI and non-infected individuals . Sera from LTBI and non-infected vaccinated individuals were diluted 1:300 and incubated with PE_PGRS33, PE, PGRS, and the control protein GST. Antigen-specific IgG (A) and IgG1 (B) were evaluated by ELISA. Letters in legend represent each individual tested and symbols correspond to OD readings. Mean values are showed as horizontal bars.

    Journal: Frontiers in Immunology

    Article Title: The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    doi: 10.3389/fimmu.2014.00236

    Figure Lengend Snippet: Antigen-specific antibody response in LTBI and non-infected individuals . Sera from LTBI and non-infected vaccinated individuals were diluted 1:300 and incubated with PE_PGRS33, PE, PGRS, and the control protein GST. Antigen-specific IgG (A) and IgG1 (B) were evaluated by ELISA. Letters in legend represent each individual tested and symbols correspond to OD readings. Mean values are showed as horizontal bars.

    Article Snippet: Viable cells were counted by trypan blue exclusion, and 1.5 million cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B (Calbiochem, Pacific Center, CA, USA) in 24-well plates at 37°C in 5% CO2 for 96 h. Polymyxin B was added to rule out the possibility of contamination with lipopolysaccharide (LPS).

    Techniques: Infection, Incubation, Enzyme-linked Immunosorbent Assay

    Humoral immune response to PE_PGRS33, PGRS, and PE in immunized mice . BALB/c mice were immunized with PE_PGRS33 and sera were collected 15 days after the last immunization. IgG (A) , IgG1 (B) , and IgG2a (C) responses to PE_PGRS33 (□), PE (▲), PGRS (○), and the control protein GST (♦), were evaluated by ELISA assay. For total IgG determination, each serum was diluted 1:100 and for IgG1 and IgG2a determination, mice sera were diluted as indicated in the figure. Bars (A) and datum points (B,C) are the average readings ± SD of data from four mice in the group, and the results showed are representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    doi: 10.3389/fimmu.2014.00236

    Figure Lengend Snippet: Humoral immune response to PE_PGRS33, PGRS, and PE in immunized mice . BALB/c mice were immunized with PE_PGRS33 and sera were collected 15 days after the last immunization. IgG (A) , IgG1 (B) , and IgG2a (C) responses to PE_PGRS33 (□), PE (▲), PGRS (○), and the control protein GST (♦), were evaluated by ELISA assay. For total IgG determination, each serum was diluted 1:100 and for IgG1 and IgG2a determination, mice sera were diluted as indicated in the figure. Bars (A) and datum points (B,C) are the average readings ± SD of data from four mice in the group, and the results showed are representative of three independent experiments.

    Article Snippet: Viable cells were counted by trypan blue exclusion, and 1.5 million cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B (Calbiochem, Pacific Center, CA, USA) in 24-well plates at 37°C in 5% CO2 for 96 h. Polymyxin B was added to rule out the possibility of contamination with lipopolysaccharide (LPS).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Antigen-specific IFN-γ secretion in immunized mice . Spleen cells from mice immunized with PE_PGRS33 or from control mice were incubated with 25 μg of PE_PGRS33, PGRS, PE, or GST recombinant purified proteins plus 10 μg/ml of polymyxin B for 4 days. The culture supernatants were evaluated for IFN-γ production using ELISA assay. Each bar represents the mean ± SD of data from four mice per group. Representative results of three independent experiments are shown.

    Journal: Frontiers in Immunology

    Article Title: The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    doi: 10.3389/fimmu.2014.00236

    Figure Lengend Snippet: Antigen-specific IFN-γ secretion in immunized mice . Spleen cells from mice immunized with PE_PGRS33 or from control mice were incubated with 25 μg of PE_PGRS33, PGRS, PE, or GST recombinant purified proteins plus 10 μg/ml of polymyxin B for 4 days. The culture supernatants were evaluated for IFN-γ production using ELISA assay. Each bar represents the mean ± SD of data from four mice per group. Representative results of three independent experiments are shown.

    Article Snippet: Viable cells were counted by trypan blue exclusion, and 1.5 million cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B (Calbiochem, Pacific Center, CA, USA) in 24-well plates at 37°C in 5% CO2 for 96 h. Polymyxin B was added to rule out the possibility of contamination with lipopolysaccharide (LPS).

    Techniques: Mouse Assay, Incubation, Recombinant, Purification, Enzyme-linked Immunosorbent Assay

    INF-γ response to the PE_PGRS33 and its PE and PGRS domains in LTBI and non-infected individuals . Diluted whole blood cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B for 6 days. IFN-γ in the supernatants was quantified using the Human IFN-γ ELISA kit provided with the QuantiFERON ® -TB Gold Kit. Letters represent each individual tested and symbols correspond to the amount of IFN-γ produced by each of them.

    Journal: Frontiers in Immunology

    Article Title: The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans

    doi: 10.3389/fimmu.2014.00236

    Figure Lengend Snippet: INF-γ response to the PE_PGRS33 and its PE and PGRS domains in LTBI and non-infected individuals . Diluted whole blood cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B for 6 days. IFN-γ in the supernatants was quantified using the Human IFN-γ ELISA kit provided with the QuantiFERON ® -TB Gold Kit. Letters represent each individual tested and symbols correspond to the amount of IFN-γ produced by each of them.

    Article Snippet: Viable cells were counted by trypan blue exclusion, and 1.5 million cells were stimulated with 25 μg of PE_PGRS33, PE, PGRS, or GST protein plus 10 μg/ml of polymyxin B (Calbiochem, Pacific Center, CA, USA) in 24-well plates at 37°C in 5% CO2 for 96 h. Polymyxin B was added to rule out the possibility of contamination with lipopolysaccharide (LPS).

    Techniques: Infection, Enzyme-linked Immunosorbent Assay, Produced

    A short region immediately C-terminal to the charged region of SF3a120 is sufficient for SF3a66 binding. A , scheme of in vitro -translated and GST-tagged SF3a120 proteins. Boxes indicate known protein domains as follows: S1 and S2 , SURP1 and SURP 2 motifs;

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction Domains and Nuclear Targeting Signals in Subunits of the U2 Small Nuclear Ribonucleoprotein Particle-associated Splicing Factor SF3a *

    doi: 10.1074/jbc.M110.201491

    Figure Lengend Snippet: A short region immediately C-terminal to the charged region of SF3a120 is sufficient for SF3a66 binding. A , scheme of in vitro -translated and GST-tagged SF3a120 proteins. Boxes indicate known protein domains as follows: S1 and S2 , SURP1 and SURP 2 motifs;

    Article Snippet: Sequences encoding GST-tagged, internal regions of SF3a120 were cloned into the BamHI/EcoRI sites of pGEX6P-2 (Amersham Biosciences).

    Techniques: Binding Assay, In Vitro

    Amino acids 145–243 of SF3a120 are sufficient for SF3a60 binding. A , scheme of in vitro -translated SF3a120 proteins. Protein domains are as defined in the legend to . B , SDS-PAGE and Coomassie Blue staining of recombinant GST ( lane 1 ) and

    Journal: The Journal of Biological Chemistry

    Article Title: Interaction Domains and Nuclear Targeting Signals in Subunits of the U2 Small Nuclear Ribonucleoprotein Particle-associated Splicing Factor SF3a *

    doi: 10.1074/jbc.M110.201491

    Figure Lengend Snippet: Amino acids 145–243 of SF3a120 are sufficient for SF3a60 binding. A , scheme of in vitro -translated SF3a120 proteins. Protein domains are as defined in the legend to . B , SDS-PAGE and Coomassie Blue staining of recombinant GST ( lane 1 ) and

    Article Snippet: Sequences encoding GST-tagged, internal regions of SF3a120 were cloned into the BamHI/EcoRI sites of pGEX6P-2 (Amersham Biosciences).

    Techniques: Binding Assay, In Vitro, SDS Page, Staining, Recombinant

    DEK40 recognizes and directly binds cox3 , nad2 , and nad5 transcripts. (A) Nucleotide sequence of RNA probes. Edited sites are indicated in red. (B) RNA EMSAs indicated that DEK40 directly binds to cox3 transcripts that surround the cox3 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. Black arrows show the shifted bound and free RNA probe. (C) RNA EMSAs indicated that DEK40 directly binds to nad2 transcripts that surround the nad2 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probesw are indicated by black arrows. (D) RNA EMSAs indicated that DEK40 directly binds to nad5 transcripts that surround the nad5 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probes are indicated by black arrows.

    Journal: Journal of Experimental Botany

    Article Title: Pentatricopeptide repeat protein DEK40 is required for mitochondrial function and kernel development in maize

    doi: 10.1093/jxb/erz391

    Figure Lengend Snippet: DEK40 recognizes and directly binds cox3 , nad2 , and nad5 transcripts. (A) Nucleotide sequence of RNA probes. Edited sites are indicated in red. (B) RNA EMSAs indicated that DEK40 directly binds to cox3 transcripts that surround the cox3 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. Black arrows show the shifted bound and free RNA probe. (C) RNA EMSAs indicated that DEK40 directly binds to nad2 transcripts that surround the nad2 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probesw are indicated by black arrows. (D) RNA EMSAs indicated that DEK40 directly binds to nad5 transcripts that surround the nad5 edited site. Unlabeled probe was used as a competitor, and GST was used as a negative control. The shifted bound and free RNA probes are indicated by black arrows.

    Article Snippet: DEK40 tagged with GST was purified using glutathione Sepharose 4B (GE Healthcare).

    Techniques: Sequencing, Negative Control

    In vitro  kinase assay with HIS tag–purified PCRan1p from  Saccharomyces cerevisiae  strain INVSC, containing pYES2.1-TOPO plus  PCRan1 , tested for phosphorylation activity with a series of ( A ) temperatures, ( B ) pH, and (C) divalent metal cations, using eukaryotic translation initiation factor 4E binding protein 1 (PHAS-I) as the substrate. ( D )  In vitro  kinase assay with glutathione S-transferase tag–purified PCRan1p and PCMei2p that were generated in  Escherichia coli  BL21(DE3)pLysS cells, and tested for similar PCRan1p temperature-dependant kinase activity as in  A  using the native substrate of PCRan1p, PCMei2p. Default conditions, unless otherwise stated in figure, are 25°C, 10 mM MnCl 2  and 10 mM MgCl 2 , and pH 7.

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity

    doi: 10.1165/rcmb.2008-0098OC

    Figure Lengend Snippet: In vitro kinase assay with HIS tag–purified PCRan1p from Saccharomyces cerevisiae strain INVSC, containing pYES2.1-TOPO plus PCRan1 , tested for phosphorylation activity with a series of ( A ) temperatures, ( B ) pH, and (C) divalent metal cations, using eukaryotic translation initiation factor 4E binding protein 1 (PHAS-I) as the substrate. ( D ) In vitro kinase assay with glutathione S-transferase tag–purified PCRan1p and PCMei2p that were generated in Escherichia coli BL21(DE3)pLysS cells, and tested for similar PCRan1p temperature-dependant kinase activity as in A using the native substrate of PCRan1p, PCMei2p. Default conditions, unless otherwise stated in figure, are 25°C, 10 mM MnCl 2 and 10 mM MgCl 2 , and pH 7.

    Article Snippet: Supernatant was removed and glutathione S-transferase–tagged PcRan1 and PcMei2 proteins were purified over glutathione-sepharose, according to manufacturer's directions (GE Life Sciences).

    Techniques: In Vitro, Kinase Assay, Purification, Activity Assay, Binding Assay, Generated

    β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Journal: The Journal of Biological Chemistry

    Article Title: Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *

    doi: 10.1074/jbc.M115.666065

    Figure Lengend Snippet: β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Article Snippet: It is known that GST alone forms dimers ( G ), but our data demonstrate that GST-A3H does not form dimers through the GST tag, or we would be unable to resolve A3H haplotype II R44A/Y46A as a monomer ( D ).

    Techniques: Size-exclusion Chromatography, Concentration Assay, Sequencing, Construct, SDS Page

    UBA domain of AMPK-related kinases do not bind tetra-ubiquitin or polyubiquitin ( A ) GST-fusion proteins of the indicated UBA domains of AMPK subfamily kinases or the tandem UBA domains of Rhp23 bound to glutathione–Sepharose were incubated with either tetra-ubiquitin or K 48 -linked polyubiquitin. The beads were washed and the associated protein was eluted and subjected to PAGE. The gels were either stained with Coomassie Blue (lower panel) to visualize levels of GST-fusion proteins or immunoblotted with anti-ubiquitin antibody to detect associated ubiquitin forms (upper panels). ( B ]. The level of GST-fusion proteins associated with the glutathione–Sepharose was assessed by immunoblot analysis employing a anti-GST antibody (lower panel). The results are representative of two or three separate experiments.

    Journal: Biochemical Journal

    Article Title: The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation

    doi: 10.1042/BJ20051844

    Figure Lengend Snippet: UBA domain of AMPK-related kinases do not bind tetra-ubiquitin or polyubiquitin ( A ) GST-fusion proteins of the indicated UBA domains of AMPK subfamily kinases or the tandem UBA domains of Rhp23 bound to glutathione–Sepharose were incubated with either tetra-ubiquitin or K 48 -linked polyubiquitin. The beads were washed and the associated protein was eluted and subjected to PAGE. The gels were either stained with Coomassie Blue (lower panel) to visualize levels of GST-fusion proteins or immunoblotted with anti-ubiquitin antibody to detect associated ubiquitin forms (upper panels). ( B ]. The level of GST-fusion proteins associated with the glutathione–Sepharose was assessed by immunoblot analysis employing a anti-GST antibody (lower panel). The results are representative of two or three separate experiments.

    Article Snippet: The beads were then washed three times with 40 ml of Buffer B containing 0.5 M NaCl, followed by three washes with Buffer C. The resin was then resuspended to a final volume of 15 ml in Buffer C. The amount of protein bound to the glutathione–Sepharose was quantified, and 50 μg of GST-tagged-PreScission™ protease (Amersham)/mg of protein bound to the resin was added.

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Staining

    Role of the UBA domain in regulating AMPK-related kinase activity and T-loop phosphorylation ( A – E ) HEK-293 cells were transfected with constructs encoding GST-fusion proteins of full-length wild type (WT) or indicated mutants of the AMPK-related kinases. ‘T/A’ indicates mutants in which the LKB1 T-loop threonine residue is changed to alanine. KD is a fragment of the enzyme encompassing only the kinase domain (MARK4 residues 2–329; SIK residues 2–305, MARK3 residues 2–326) and KD-UBA is a fragment that encompasses the kinase domain and UBA domains MARK4 residues 2–400, SIK residues 2–350 and MARK3 residues 2–383). At 36 h post-transfection, the AMPK-related kinases were affinity-purified from the cell lysates using glutathione–Sepharose and assayed for AMARA-peptide kinase activity (top panel) or subjected to immunoblot analysis with an anti-GST antibody to quantify relative protein levels (Total) or immunoblotted with T-loop phosphoantibodies recognizing the LKB1 phosphorylated forms of these enzymes (Phospho-T-loop). Results of the kinase catalytic assays are presented as the mean catalytic activities±S.D. for assays carried out in triplicate. The results presented are representative of two or three independent experiments.

    Journal: Biochemical Journal

    Article Title: The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation

    doi: 10.1042/BJ20051844

    Figure Lengend Snippet: Role of the UBA domain in regulating AMPK-related kinase activity and T-loop phosphorylation ( A – E ) HEK-293 cells were transfected with constructs encoding GST-fusion proteins of full-length wild type (WT) or indicated mutants of the AMPK-related kinases. ‘T/A’ indicates mutants in which the LKB1 T-loop threonine residue is changed to alanine. KD is a fragment of the enzyme encompassing only the kinase domain (MARK4 residues 2–329; SIK residues 2–305, MARK3 residues 2–326) and KD-UBA is a fragment that encompasses the kinase domain and UBA domains MARK4 residues 2–400, SIK residues 2–350 and MARK3 residues 2–383). At 36 h post-transfection, the AMPK-related kinases were affinity-purified from the cell lysates using glutathione–Sepharose and assayed for AMARA-peptide kinase activity (top panel) or subjected to immunoblot analysis with an anti-GST antibody to quantify relative protein levels (Total) or immunoblotted with T-loop phosphoantibodies recognizing the LKB1 phosphorylated forms of these enzymes (Phospho-T-loop). Results of the kinase catalytic assays are presented as the mean catalytic activities±S.D. for assays carried out in triplicate. The results presented are representative of two or three independent experiments.

    Article Snippet: The beads were then washed three times with 40 ml of Buffer B containing 0.5 M NaCl, followed by three washes with Buffer C. The resin was then resuspended to a final volume of 15 ml in Buffer C. The amount of protein bound to the glutathione–Sepharose was quantified, and 50 μg of GST-tagged-PreScission™ protease (Amersham)/mg of protein bound to the resin was added.

    Techniques: Activity Assay, Transfection, Construct, Affinity Purification

    ARF6 is regulated differently in CNS vs . PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of fluoresent signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (+DIV10) labelled with GGA3-ABD-GST to detect active ARF. Graph shows quantification of ARF activation in the two axon types. n=58(DRG) and 60(cortical). ***p

    Journal: bioRxiv

    Article Title: EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

    doi: 10.1101/150037

    Figure Lengend Snippet: ARF6 is regulated differently in CNS vs . PNS neurons. (A) Cortical neurons and adult DRG neurons immunolabelled for EFA6 (upper panels) or ACAP1 (lower panels). Both neuronal types were labelled and imaged identically to allow comparison of fluoresent signal. Images represent two independent immunolabelling experiments. (B) Axons of DRG and cortical neurons (+DIV10) labelled with GGA3-ABD-GST to detect active ARF. Graph shows quantification of ARF activation in the two axon types. n=58(DRG) and 60(cortical). ***p

    Article Snippet: Axonal ARF activation assayActive ARF was detected using a peptide derived from the active ARF binding domain (ABD) of GGA3 fused to a GST tag (GGA3-ABD- GST, Thermo).

    Techniques: Activation Assay

    EFA6 activates ARF throughout mature CNS axons. (A) Active ARF (GGA3-ABD-GST, green) in proximal axons (neurofascin in blue), and distal axons (axon neurofilaments, red). Ellipse indicates the initial segment, arrows indicate a distal axon. (B) In young neurons (+4DIV), active ARF is detected in sparse tubulo-vesicular structures throughout developing axons which diminish at the growth cone. (C) In differentiated neurons (+14DIV) active ARF (GGA3-ABD-GST + anti-GST) is distributed uniformly throughout axons (as indicated by immunolabelling with SMI312 for axonal neurofilaments). (D) Active ARF and total ARF6 in axons of neurons expressing either control shRNA or shRNA targeting EFA6 (red). Active ARF or ARF6 is shown in green, as indicated. Arrows indicate axons. Quantification of axonal ARF activity and total ARF6 in neurons expressing either control or EFA6 shRNA. n=61 to 64 neurons (active ARF), *** p

    Journal: bioRxiv

    Article Title: EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment

    doi: 10.1101/150037

    Figure Lengend Snippet: EFA6 activates ARF throughout mature CNS axons. (A) Active ARF (GGA3-ABD-GST, green) in proximal axons (neurofascin in blue), and distal axons (axon neurofilaments, red). Ellipse indicates the initial segment, arrows indicate a distal axon. (B) In young neurons (+4DIV), active ARF is detected in sparse tubulo-vesicular structures throughout developing axons which diminish at the growth cone. (C) In differentiated neurons (+14DIV) active ARF (GGA3-ABD-GST + anti-GST) is distributed uniformly throughout axons (as indicated by immunolabelling with SMI312 for axonal neurofilaments). (D) Active ARF and total ARF6 in axons of neurons expressing either control shRNA or shRNA targeting EFA6 (red). Active ARF or ARF6 is shown in green, as indicated. Arrows indicate axons. Quantification of axonal ARF activity and total ARF6 in neurons expressing either control or EFA6 shRNA. n=61 to 64 neurons (active ARF), *** p

    Article Snippet: Axonal ARF activation assayActive ARF was detected using a peptide derived from the active ARF binding domain (ABD) of GGA3 fused to a GST tag (GGA3-ABD- GST, Thermo).

    Techniques: Expressing, shRNA, Activity Assay

    LegK2 interacts with ARPC1B and ARP3 subunits of the ARP2/3 complex. (A) Yeast two-hybrid assays of L. pneumophila protein kinase LegK1/LegK2 and ARPC1B/ARP3 subunits of the human ARP2/3 complex. Diploids from the mating of AH109(pGBKT7-legK1) or AH109(pGBKT7-legK2) with Y187(pACT2), Y187(pACT2-ARPC1B), or Y187(pACT2-ACTR3) were grown on SD medium without tryptophan (−W) or leucine (−L). Histidine auxotrophy was tested by plating 5 or 50 µl of diploids on SD-W-L medium without histidine (−H) in the presence of 10 mM 3-AT. (B) Affinity copurification of Flag-tagged ARP2/3 subunits with GST-tagged LegK2 protein. HEK293T cells were cotransfected with pDEST27, pDEST27-legK2, or pDEST27-legK2 K112M and pCI-Neo3Flag-ARPC1B or -ACTR3. GST, GST-tagged LegK2, or LegK2 K112M was purified on glutathione-agarose 4B, and purified fractions were immunoblotted with both anti-GST and anti-Flag antibodies. (C) Cellular localization of ARPC1B/ARP3 and LegK2/LegK1 proteins in HEK293T cells cotransfected with pDEST27, pDEST27-legK2, or -legK1 and pCI-Neo3Flag-ARPC1B or -ACTR3. The GST, GST-LegK2, and GST-LegK1 proteins were detected by immunofluorescence with anti-GST antibodies (green), and 3Flag-ARPC1B and 3Flag-ARP3 were detected with anti-Flag antibodies (red). Scale bars, 10 µm. (D) Quantitation of colocalization by Pearson coefficient. The Pearson coefficient was determined with the JACoP plugin of the ImageJ software and is expressed as the mean value calculated for 30 cells.

    Journal: mBio

    Article Title: The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

    doi: 10.1128/mBio.00354-15

    Figure Lengend Snippet: LegK2 interacts with ARPC1B and ARP3 subunits of the ARP2/3 complex. (A) Yeast two-hybrid assays of L. pneumophila protein kinase LegK1/LegK2 and ARPC1B/ARP3 subunits of the human ARP2/3 complex. Diploids from the mating of AH109(pGBKT7-legK1) or AH109(pGBKT7-legK2) with Y187(pACT2), Y187(pACT2-ARPC1B), or Y187(pACT2-ACTR3) were grown on SD medium without tryptophan (−W) or leucine (−L). Histidine auxotrophy was tested by plating 5 or 50 µl of diploids on SD-W-L medium without histidine (−H) in the presence of 10 mM 3-AT. (B) Affinity copurification of Flag-tagged ARP2/3 subunits with GST-tagged LegK2 protein. HEK293T cells were cotransfected with pDEST27, pDEST27-legK2, or pDEST27-legK2 K112M and pCI-Neo3Flag-ARPC1B or -ACTR3. GST, GST-tagged LegK2, or LegK2 K112M was purified on glutathione-agarose 4B, and purified fractions were immunoblotted with both anti-GST and anti-Flag antibodies. (C) Cellular localization of ARPC1B/ARP3 and LegK2/LegK1 proteins in HEK293T cells cotransfected with pDEST27, pDEST27-legK2, or -legK1 and pCI-Neo3Flag-ARPC1B or -ACTR3. The GST, GST-LegK2, and GST-LegK1 proteins were detected by immunofluorescence with anti-GST antibodies (green), and 3Flag-ARPC1B and 3Flag-ARP3 were detected with anti-Flag antibodies (red). Scale bars, 10 µm. (D) Quantitation of colocalization by Pearson coefficient. The Pearson coefficient was determined with the JACoP plugin of the ImageJ software and is expressed as the mean value calculated for 30 cells.

    Article Snippet: Purified fractions were immunoblotted with both anti-GST (13-6700; Invitrogen) and anti-Flag (clone M2; Sigma) antibodies to detect GST or GST-tagged LegK2 protein and Flag-tagged ARP2/3 subunits, respectively.

    Techniques: Copurification, Purification, Immunofluorescence, Quantitation Assay, Software

    Mad2 is a new substrate for CCP6 in MKs. (A) Protein polyglutamylation was assessed by immunoblotting with GT335 antibody. Spleen and BM lysates from CCP6 -deficient and littermate control mice were analyzed. Data were repeated three times with similar results. (B) Recombinant CCP6-wt and inactive CCP6 mutant (CCP6-mut) were immobilized with Affi-gel10 resin, to which mouse BM lysates were added for affinity chromatography. The eluted fractions were visualized by SDS-PAGE, followed by silver staining. M: molecular weight marker. The differential band of 30 kD seen in CCP6-mut gel was cut for mass spectrometry and identified as Mad2. The peptide sequences and coverage analyzed by LC-LTQ MS/MS are shown in the bottom graph. (C) Glutamylated rGST-Mad2 binding to CCP6-mut protein was analyzed by GST pulldown. rGST-Mad2 protein was incubated with lysates from Myc-tagged CCP6-wt or Myc-tagged CCP6-mut expressed 293T cells at 37°C for 2 h, followed by incubation with GST beads. Ratios of pulldown/input of CCP6 were calculated and shown as means ± SD. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Cytosolic carboxypeptidase CCP6 is required for megakaryopoiesis by modulating Mad2 polyglutamylation

    doi: 10.1084/jem.20141123

    Figure Lengend Snippet: Mad2 is a new substrate for CCP6 in MKs. (A) Protein polyglutamylation was assessed by immunoblotting with GT335 antibody. Spleen and BM lysates from CCP6 -deficient and littermate control mice were analyzed. Data were repeated three times with similar results. (B) Recombinant CCP6-wt and inactive CCP6 mutant (CCP6-mut) were immobilized with Affi-gel10 resin, to which mouse BM lysates were added for affinity chromatography. The eluted fractions were visualized by SDS-PAGE, followed by silver staining. M: molecular weight marker. The differential band of 30 kD seen in CCP6-mut gel was cut for mass spectrometry and identified as Mad2. The peptide sequences and coverage analyzed by LC-LTQ MS/MS are shown in the bottom graph. (C) Glutamylated rGST-Mad2 binding to CCP6-mut protein was analyzed by GST pulldown. rGST-Mad2 protein was incubated with lysates from Myc-tagged CCP6-wt or Myc-tagged CCP6-mut expressed 293T cells at 37°C for 2 h, followed by incubation with GST beads. Ratios of pulldown/input of CCP6 were calculated and shown as means ± SD. **, P

    Article Snippet: PercP-CY5.5 goat anti–rat IgG and the antibodies against myc-tag, Mad2, CCP6, and GST-tag were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Mouse Assay, Recombinant, Mutagenesis, Affinity Chromatography, SDS Page, Silver Staining, Molecular Weight, Marker, Mass Spectrometry, Binding Assay, Incubation

    Densitometric analysis of westerns blots of total p38 MAPK expression in hearts from wild-type ( WT ) and knockout (KO) mice, immunoblotted against known quantities of GST-tagged recombinant p38 MAPK ( n = 6/recombinant protein or heart sample). Error bars represent SEM. A representative western blot is shown in the lower panel

    Journal: Cell Stress & Chaperones

    Article Title: MAPKAPK-2 modulates p38-MAPK localization and small heat shock protein phosphorylation but does not mediate the injury associated with p38-MAPK activation during myocardial ischemia

    doi: 10.1007/s12192-009-0101-5

    Figure Lengend Snippet: Densitometric analysis of westerns blots of total p38 MAPK expression in hearts from wild-type ( WT ) and knockout (KO) mice, immunoblotted against known quantities of GST-tagged recombinant p38 MAPK ( n = 6/recombinant protein or heart sample). Error bars represent SEM. A representative western blot is shown in the lower panel

    Article Snippet: Since total p38 MAPK is markedly reduced in MK2−/− compared to MK2+/+ hearts, total p38 MAPK was quantitated by immunoblotting baseline samples of ventricular tissue (MK2−/− , n = 6 and MK2+/+ , n = 6) against known quantities of GST-tagged (Sigma, UK) recombinant p38 MAPK as shown in Fig. .

    Techniques: Expressing, Knock-Out, Mouse Assay, Recombinant, Western Blot

    A schematic model for the function of the L11-MDM2-p53 pathway. (See text for discussion.)

    Journal: Molecular and Cellular Biology

    Article Title: Ribosomal Protein L11 Negatively Regulates Oncoprotein MDM2 and Mediates a p53-Dependent Ribosomal-Stress Checkpoint Pathway

    doi: 10.1128/MCB.23.23.8902-8912.2003

    Figure Lengend Snippet: A schematic model for the function of the L11-MDM2-p53 pathway. (See text for discussion.)

    Article Snippet: Affinity-purified rabbit polyclonal antibody to human MDM2 (N-20; Santa Cruz), goat polyclonal antibody to human p53 (FL393; Santa Cruz), mouse monoclonal antibodies to p53 (clone PAb421; Oncogene Science, Uniondale, N.Y.), human MDM2 (clone SMP14; NeoMarkers), tubulin (clone DM1A + DM1B; NeoMarkers), and actin (Sigma) were purchased commercially.

    Techniques:

    Inhibitory effects of NTZ on the HBx–DDB1 interaction in vitro. GST-tagged recombinant DDB1 protein and untagged recombinant HBx protein were mixed in vitro. NTZ or DMSO was added to the mixture and incubated for 20 minutes, followed by pull-down using anti-GST antibody and Western blotting to determine the levels of DDB1 and HBx in the pulled-down samples. Representative results of 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; * P = 7.1 × 10 –5 ( t test).

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx–DDB1 Interaction

    doi: 10.1016/j.jcmgh.2018.10.010

    Figure Lengend Snippet: Inhibitory effects of NTZ on the HBx–DDB1 interaction in vitro. GST-tagged recombinant DDB1 protein and untagged recombinant HBx protein were mixed in vitro. NTZ or DMSO was added to the mixture and incubated for 20 minutes, followed by pull-down using anti-GST antibody and Western blotting to determine the levels of DDB1 and HBx in the pulled-down samples. Representative results of 3 independent experiments are shown. Summarized results of relative band intensities are shown below the panels (n = 3). NS, not significant; * P = 7.1 × 10 –5 ( t test).

    Article Snippet: The following antibodies were used: Smc5 (#ab18038, 1:10,000 [Abcam, Cambridge, United Kingdom]), β-actin (#A1978, 1:10,000 [Sigma-Aldrich, St. Louis, MO] or #5125, 1:10,000 [Cell Signaling Technology, Danvers, MA]), Hepatitis B preS2 (#NB100-93580, 1:1000 [Novus Biologicals, Littleton, CO]), DDB1 and GST tag ((#5428, 1:1000 and #2625S, 1:1000, respectively [Cell Signaling Technology]), and Flag (DYKDDDDK) tag (#018-22381, 1:1000 [Wako Pure Chemical Industries]).

    Techniques: In Vitro, Recombinant, Incubation, Western Blot

    Annexin A2 expression in ovarian cancer cell lines, peritoneal cell line and co-cultured ovarian cancer and peritoneal cells (A) Annexin A2 expression in OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9 cell lines determined by real-time PCR and were assessed using 2 −ΔΔCT quantitation method. Data represents triplicate determinations ± SEM from 2 independent experiments. (B) Western immunoblotting shows annexin A2 band at 37 kDa in cell lysates of OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9. β-actin was used as loading control. (C) Western immunoblotting shows two isoforms of annexin A2 at 37 kDa and 36 kDa bands in the conditioned media (CM) of LP-9 cells alone and a 35 kDa annexin A2 band present in the CM of co-cultured LP-9 and ovarian cancer cells. (D) 2D-western immunoblotting showed multiple annexin A2 spots at 37 kDa in the CM of LP-9 cells alone and annexin A2 isoforms at 37 kDa and 35 kDa in the CM of co-cultured LP-9 and OVCAR-5 cells over a pI values range of 6 to 8. (E) Annexin A2 western immunoblotting of recombinant annexin A2 with GST tag (~63.4 kDa) and a cleaved annexin A2 at ~36 kDa in the presence of plasmin that was partially inhibited by α2-antiplasmin. (F) Annexin A2 western immunoblotting of the CM of co-cultured OVCAR-5 and LP-9 cells treated with α2-antiplasmin, protease inhibitor cocktail, ε-aminocaproic acid (ε-ACA), GM-6001 and DMSO.

    Journal: Oncotarget

    Article Title: Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis

    doi:

    Figure Lengend Snippet: Annexin A2 expression in ovarian cancer cell lines, peritoneal cell line and co-cultured ovarian cancer and peritoneal cells (A) Annexin A2 expression in OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9 cell lines determined by real-time PCR and were assessed using 2 −ΔΔCT quantitation method. Data represents triplicate determinations ± SEM from 2 independent experiments. (B) Western immunoblotting shows annexin A2 band at 37 kDa in cell lysates of OV-90, SKOV-3, OVCAR-5, OVCAR-3 and LP-9. β-actin was used as loading control. (C) Western immunoblotting shows two isoforms of annexin A2 at 37 kDa and 36 kDa bands in the conditioned media (CM) of LP-9 cells alone and a 35 kDa annexin A2 band present in the CM of co-cultured LP-9 and ovarian cancer cells. (D) 2D-western immunoblotting showed multiple annexin A2 spots at 37 kDa in the CM of LP-9 cells alone and annexin A2 isoforms at 37 kDa and 35 kDa in the CM of co-cultured LP-9 and OVCAR-5 cells over a pI values range of 6 to 8. (E) Annexin A2 western immunoblotting of recombinant annexin A2 with GST tag (~63.4 kDa) and a cleaved annexin A2 at ~36 kDa in the presence of plasmin that was partially inhibited by α2-antiplasmin. (F) Annexin A2 western immunoblotting of the CM of co-cultured OVCAR-5 and LP-9 cells treated with α2-antiplasmin, protease inhibitor cocktail, ε-aminocaproic acid (ε-ACA), GM-6001 and DMSO.

    Article Snippet: For the plasmin digestion, recombinant annexin A2 protein with a GST-tag (0.5 μg, Abnova, Taiwan) was incubated with plasmin (0.2 U/ml, Sigma-Aldrich) for 3 h at 37oC in the presence and absence of α2-antiplasmin (0.2 μM, Calbiochem).

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitation Assay, Western Blot, Recombinant, Protease Inhibitor

    G3BP1 and G3BP2 interact with thousands of transcripts. ( a ) The G3BP1 domain structure. ( b ) GST western blot showing RNA-sequence-context-dependent inhibition of GST-G3BP1 binding to RNA by m 6 A. Molecular weights are indicated on the right. ( c , d ) RNA cross-link immunoprecipitation in HEK293 cells expressing either Flag-G3BP1 or Flag-G3BP2. Flag western blots ( c ) and radioactively labeled RNA-protein complexes ( d ) are shown. OE, overexpression; Ctrl, control. ( e ) Common peaks between PAR-CLIP biological replicates 1 and 2 for G3BP1 (top left) and G3BP2 (top right), and comparisons between G3BP1 and G3BP2 peaks (bottom left) and target genes (bottom right). ( f ) Metagene profiles of G3BP1 and G3BP2 distribution across the transcriptome. ( g ) The most enriched consensus sequences of G3BP1 (top) and G3BP2 (bottom) on mRNA; P values were determined as described in the Online Methods. ( h ) The most significantly enriched GO terms among G3BP1 target genes. The numbers to the right of the bars represent the number of genes in that GO term. ( i ) The number of G3BP1 and G3BP2 peaks that overlap with m 6 A peaks among m 6 A-containing mRNAs. ( j ) The number of G3BP1 and G3BP2 peaks on m 6 A-containing mRNAs that overlap with m 6 A. ( k ) The distribution of G3BP1 (left) and G3BP2 (right) relative to m 6 . The distance between PAR-CLIP peaks and m 6 A sites was counted in 10-nucleotide bins. A Gaussian kernel smoothing over the histogram is plotted as a transparent black line. The uncropped blot image for b . Source data for i and j .

    Journal: Nature structural & molecular biology

    Article Title: N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis

    doi: 10.1038/nsmb.3462

    Figure Lengend Snippet: G3BP1 and G3BP2 interact with thousands of transcripts. ( a ) The G3BP1 domain structure. ( b ) GST western blot showing RNA-sequence-context-dependent inhibition of GST-G3BP1 binding to RNA by m 6 A. Molecular weights are indicated on the right. ( c , d ) RNA cross-link immunoprecipitation in HEK293 cells expressing either Flag-G3BP1 or Flag-G3BP2. Flag western blots ( c ) and radioactively labeled RNA-protein complexes ( d ) are shown. OE, overexpression; Ctrl, control. ( e ) Common peaks between PAR-CLIP biological replicates 1 and 2 for G3BP1 (top left) and G3BP2 (top right), and comparisons between G3BP1 and G3BP2 peaks (bottom left) and target genes (bottom right). ( f ) Metagene profiles of G3BP1 and G3BP2 distribution across the transcriptome. ( g ) The most enriched consensus sequences of G3BP1 (top) and G3BP2 (bottom) on mRNA; P values were determined as described in the Online Methods. ( h ) The most significantly enriched GO terms among G3BP1 target genes. The numbers to the right of the bars represent the number of genes in that GO term. ( i ) The number of G3BP1 and G3BP2 peaks that overlap with m 6 A peaks among m 6 A-containing mRNAs. ( j ) The number of G3BP1 and G3BP2 peaks on m 6 A-containing mRNAs that overlap with m 6 A. ( k ) The distribution of G3BP1 (left) and G3BP2 (right) relative to m 6 . The distance between PAR-CLIP peaks and m 6 A sites was counted in 10-nucleotide bins. A Gaussian kernel smoothing over the histogram is plotted as a transparent black line. The uncropped blot image for b . Source data for i and j .

    Article Snippet: Primary antibodies to the following proteins were used: G3BP1 (Santa Cruz Biotechnology; sc-98561), Flag (Sigma-Aldrich; F1804), GST (Thermo; MA4-004) and METTL3 (Bethyl Laboratories; A301-567-A).

    Techniques: Western Blot, Sequencing, Inhibition, Binding Assay, Immunoprecipitation, Expressing, Labeling, Over Expression, Cross-linking Immunoprecipitation

    FMR1 preferentially binds to m 6 A-containing mRNA in vitro and in vivo and affects the translation of its targets. ( a ) Western blots showing preferential m 6 A binding by recombinant GST-FMR1 in vitro . ( b ) Quantification of FMR1 binding to control and m 6 A probes (data are shown as mean ± s.e.m.; n = 3 independent experiments). ( c ) Predominant consensus sequences for m 6 ). ( d ) Visualization of binding of FMR1 isoforms 1 and 7 to mRNA relative to known m 6 . Randomized peaks were generated from the transcriptome and used as a control as described in the Online Methods. ( e ) Immunofluorescence analysis of the expression of Flag-HA (FH)-tagged FMR1 isoform 1 and Flag-HA-tagged FMR1 isoform 1 I304N mutant (mut) in HEK293T cells. ( f ) RNA cross-linking and immunoprecipitation in lysates from HEK293 cells expressing either Flag-HA-tagged FMR1 or Flag-HA-tagged FMR1-I304N, using anti-Flag. IP, immunoprecipitate; FT, flow-through; M, molecular weight ladder. ( g ) Representative LC-MS quantification showing enrichment of m 6 A in Flag-HA-tagged FMR1–bound mRNA. Error bars represent the range; n = 2 independent experiments. ( h . ( i ) A schematic representation of the pulsed SILAC workflow. Dox, doxycycline. ( j ) Western blots showing the results of METTL3 knockdown and inducible FMR1/FMR1 mutant protein expression in HeLa cells. Ponceau-stained protein bands are shown as loading controls. ( k ) Representative peptide spectra showing SILAC label incorporation for a peptide belonging to a protein whose translation rate is not affected by FMR1 overexpression (left) and a peptide belonging to a protein that is translationally repressed by FMR1 overexpression (right) (compare the incorporation of medium-heavy (M) and heavy (H) label in the two plots). ( l . Uncropped blot images for a , f and j . Source data for g are available online.

    Journal: Nature structural & molecular biology

    Article Title: N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis

    doi: 10.1038/nsmb.3462

    Figure Lengend Snippet: FMR1 preferentially binds to m 6 A-containing mRNA in vitro and in vivo and affects the translation of its targets. ( a ) Western blots showing preferential m 6 A binding by recombinant GST-FMR1 in vitro . ( b ) Quantification of FMR1 binding to control and m 6 A probes (data are shown as mean ± s.e.m.; n = 3 independent experiments). ( c ) Predominant consensus sequences for m 6 ). ( d ) Visualization of binding of FMR1 isoforms 1 and 7 to mRNA relative to known m 6 . Randomized peaks were generated from the transcriptome and used as a control as described in the Online Methods. ( e ) Immunofluorescence analysis of the expression of Flag-HA (FH)-tagged FMR1 isoform 1 and Flag-HA-tagged FMR1 isoform 1 I304N mutant (mut) in HEK293T cells. ( f ) RNA cross-linking and immunoprecipitation in lysates from HEK293 cells expressing either Flag-HA-tagged FMR1 or Flag-HA-tagged FMR1-I304N, using anti-Flag. IP, immunoprecipitate; FT, flow-through; M, molecular weight ladder. ( g ) Representative LC-MS quantification showing enrichment of m 6 A in Flag-HA-tagged FMR1–bound mRNA. Error bars represent the range; n = 2 independent experiments. ( h . ( i ) A schematic representation of the pulsed SILAC workflow. Dox, doxycycline. ( j ) Western blots showing the results of METTL3 knockdown and inducible FMR1/FMR1 mutant protein expression in HeLa cells. Ponceau-stained protein bands are shown as loading controls. ( k ) Representative peptide spectra showing SILAC label incorporation for a peptide belonging to a protein whose translation rate is not affected by FMR1 overexpression (left) and a peptide belonging to a protein that is translationally repressed by FMR1 overexpression (right) (compare the incorporation of medium-heavy (M) and heavy (H) label in the two plots). ( l . Uncropped blot images for a , f and j . Source data for g are available online.

    Article Snippet: Primary antibodies to the following proteins were used: G3BP1 (Santa Cruz Biotechnology; sc-98561), Flag (Sigma-Aldrich; F1804), GST (Thermo; MA4-004) and METTL3 (Bethyl Laboratories; A301-567-A).

    Techniques: In Vitro, In Vivo, Western Blot, Binding Assay, Recombinant, Generated, Immunofluorescence, Expressing, Mutagenesis, Immunoprecipitation, Flow Cytometry, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Staining, Over Expression

    ABI1 inhibits MKKK18 activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf GST–ABI1 or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The 32 P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t -test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro . GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [ 32 P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.

    Journal: Plant and Cell Physiology

    Article Title: Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

    doi: 10.1093/pcp/pcv146

    Figure Lengend Snippet: ABI1 inhibits MKKK18 activity. (A) Phosphatase activity of recombinant ABI1 and ABI2 proteins. The enzyme reactions were performed in a 50 µl final volume containing 3–5 µg pf GST–ABI1 or GST–ABI2. The results presented are the means from three independent biological replicates. (B) ABI1 inactivates MKKK18. The MKKK18–GFP immunocomplex was incubated with 3 µg of GST–ABI1 and GST–ABI2 (as a negative control), after which the kinase activity was determined with MBP as a substrate. Equal loading was confirmed by Coomassie Brilliant Blue (CBB) staining of MBP. The 32 P-labeled MBP bands were quantified and then normalized against the intensity of the corresponding control band using ImageJ software. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes determined using Student’s t -test. (C) Recombinant MKKK18 has no autophosphorylation activity in vitro . GST–MKKK18 was incubated with MBP or/and MKK3, without or in the presence of [ 32 P]ATP. Bands of GST–MKKK18, MKK3–GST and GST–SnRK2.6 are indicated by a filled circle, a triangle and an arrow, respectively. Protein loading was confirmed by CBB staining. The blots shown are representative of three independent trials.

    Article Snippet: The membranes were blocked for 1 h in PBS-T containing a 3% blocking solution, washed three times and incubated for 1 h with rabbit anti-GFP (1 : 200; sc-8334, Santa Cruz Biotechnology), StrepMAB-Classic (1 : 3,000; IBA BioTAGnology), anti-GST-Tag (1 : 5,000; Sigma), anti-MKKK18 antibody (AS13 2673, Agrisera), anti-ABI1 (AS12 1861, Agrisera) and anti-ABI2 (AS12 1871, Agrisera).

    Techniques: Activity Assay, Recombinant, Incubation, Negative Control, Staining, Labeling, Software, In Vitro

    MKKK18 interacts with the ABI1 protein phosphatase. (A) Yeast two-hybrid analysis of the interaction between MKKK18 and ABI1/2 PP2Cs. Diploid yeast colonies were grown on double (DDO-SD medium without Leu and Trp) or quadruple selective medium (QDO-SD medium without Leu, Trp, His or Ade) with or without supplemented X-α-Gal and aureobasidin. The bait (MKKK18) did not autoactivate the reporter genes in yeast. (B) Detection of ABI1/2 and MKKK18 expression in diploid yeast strains. Expression of BD and AD fusion proteins in yeast was determined by immunoblotting using specific anti-ABI1, anti-ABI2 and anti-MKKK18 antibodies. (C and D) Pull-down assays to verify the interaction of ABI1/2 with MKKK18. Input lines represent 100% of the ABI1/2. Recombinant GST–MKKK18 or His-MKKK18 was pre-coupled to glutathione–Sepharose, and incubated with StrepTag-ABI1 or StrepTag-ABI2, respectively. Pulled-down StrepTagged ABI1 protein was detected (IB) with the epitope tag antibody. The presence of recombinant protein was confirmed using anti-MKKK18 antibody. (E and F) ABI1–MKKK18 interaction occurs within the nucleus. BiFC analysis in Arabidopsis protoplasts expressing full-length ABI1/2 and MKKK18 fused to cECFP or nVenus, respectively. RFP (E) was used as a transformation control. CFP–CBP20 (cyan fluorescent protein–Cap Binding Protein 20) (F) was used as a marker of nuclear localization.

    Journal: Plant and Cell Physiology

    Article Title: Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

    doi: 10.1093/pcp/pcv146

    Figure Lengend Snippet: MKKK18 interacts with the ABI1 protein phosphatase. (A) Yeast two-hybrid analysis of the interaction between MKKK18 and ABI1/2 PP2Cs. Diploid yeast colonies were grown on double (DDO-SD medium without Leu and Trp) or quadruple selective medium (QDO-SD medium without Leu, Trp, His or Ade) with or without supplemented X-α-Gal and aureobasidin. The bait (MKKK18) did not autoactivate the reporter genes in yeast. (B) Detection of ABI1/2 and MKKK18 expression in diploid yeast strains. Expression of BD and AD fusion proteins in yeast was determined by immunoblotting using specific anti-ABI1, anti-ABI2 and anti-MKKK18 antibodies. (C and D) Pull-down assays to verify the interaction of ABI1/2 with MKKK18. Input lines represent 100% of the ABI1/2. Recombinant GST–MKKK18 or His-MKKK18 was pre-coupled to glutathione–Sepharose, and incubated with StrepTag-ABI1 or StrepTag-ABI2, respectively. Pulled-down StrepTagged ABI1 protein was detected (IB) with the epitope tag antibody. The presence of recombinant protein was confirmed using anti-MKKK18 antibody. (E and F) ABI1–MKKK18 interaction occurs within the nucleus. BiFC analysis in Arabidopsis protoplasts expressing full-length ABI1/2 and MKKK18 fused to cECFP or nVenus, respectively. RFP (E) was used as a transformation control. CFP–CBP20 (cyan fluorescent protein–Cap Binding Protein 20) (F) was used as a marker of nuclear localization.

    Article Snippet: The membranes were blocked for 1 h in PBS-T containing a 3% blocking solution, washed three times and incubated for 1 h with rabbit anti-GFP (1 : 200; sc-8334, Santa Cruz Biotechnology), StrepMAB-Classic (1 : 3,000; IBA BioTAGnology), anti-GST-Tag (1 : 5,000; Sigma), anti-MKKK18 antibody (AS13 2673, Agrisera), anti-ABI1 (AS12 1861, Agrisera) and anti-ABI2 (AS12 1871, Agrisera).

    Techniques: Expressing, Recombinant, Incubation, Bimolecular Fluorescence Complementation Assay, Transformation Assay, Binding Assay, Marker

    ABA induces rapid MKKK18 activation in tobacco. (A–C) Generation of MKKK18-specific antibodies. (A) MKKK18 peptide competition assay. Recombinant GST–MKKK18 protein was incubated with anti-MKKK18 with and without 45 µg of blocking peptide. A single band of approximatley 65 kDa specific to GST–MKKK18 is absent in the immunoprecipitates containing the blocking peptide. Immunodetection was performed using anti-MKKK18 antibody. (B) MKKK18–GFP protein immunoprecipitated from tobacco total protein extracts using increasing amounts of anti-MKKK18 and anti-GFP antibodies. The MKKK18–GFP protein was precipitated by 3 µl (lane 1) or 10 µl (lane 2) of MKKK18 antiserum and 0.6 µg (lane 3) or 1.2 µg (lane 4) of anti-GFP antibody, respectively. Immunoblotting with anti-MKKK18 antibodies confirmed the presence of MKKK18–GFP fusion protein. Arrows/Ab indicate anti-MKKK18 antibodies (visible as a band in lane 2) where excess anti-MKKK18 antibody was used in the immunoprecipitation reaction. The 65 kDa band represents MKKK18–GFP protein. (C) Analysis of MKKK18 activity in the mkkk18 knockout lines. MKKK18 protein was immunoprecipitated from 600 µg of total protein extract isolated from ABA-treated WT Col-0, mkkk18-1 , mkkk18-2 and MKKK18oe using specific anti-MKKK18 antibodies. Immunocomplex activity was determined using MBP (2 µg) as a substrate. Coomassie Brilliant Blue (CBB) staining of MBP confirmed equal loading. (D) Tobacco plants infiltrated with Agrobacterium strain C58C1 harboring 35S:MKKK18-GFP and 35S:p19 constructs for transient expression. Four to five days after infiltration, the tobacco plants were treated with ABA and tissue samples were collected at the indicated time points. MKKK18 activity was assessed by the immunocomplex assay using anti-MKKK18 antibody and MBP as a substrate. MKKK18–GFP was detected with anti-GFP antibody. CBB staining of MBP confirmed equal loading. The above experiments were repeated several times with similar results.

    Journal: Plant and Cell Physiology

    Article Title: Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

    doi: 10.1093/pcp/pcv146

    Figure Lengend Snippet: ABA induces rapid MKKK18 activation in tobacco. (A–C) Generation of MKKK18-specific antibodies. (A) MKKK18 peptide competition assay. Recombinant GST–MKKK18 protein was incubated with anti-MKKK18 with and without 45 µg of blocking peptide. A single band of approximatley 65 kDa specific to GST–MKKK18 is absent in the immunoprecipitates containing the blocking peptide. Immunodetection was performed using anti-MKKK18 antibody. (B) MKKK18–GFP protein immunoprecipitated from tobacco total protein extracts using increasing amounts of anti-MKKK18 and anti-GFP antibodies. The MKKK18–GFP protein was precipitated by 3 µl (lane 1) or 10 µl (lane 2) of MKKK18 antiserum and 0.6 µg (lane 3) or 1.2 µg (lane 4) of anti-GFP antibody, respectively. Immunoblotting with anti-MKKK18 antibodies confirmed the presence of MKKK18–GFP fusion protein. Arrows/Ab indicate anti-MKKK18 antibodies (visible as a band in lane 2) where excess anti-MKKK18 antibody was used in the immunoprecipitation reaction. The 65 kDa band represents MKKK18–GFP protein. (C) Analysis of MKKK18 activity in the mkkk18 knockout lines. MKKK18 protein was immunoprecipitated from 600 µg of total protein extract isolated from ABA-treated WT Col-0, mkkk18-1 , mkkk18-2 and MKKK18oe using specific anti-MKKK18 antibodies. Immunocomplex activity was determined using MBP (2 µg) as a substrate. Coomassie Brilliant Blue (CBB) staining of MBP confirmed equal loading. (D) Tobacco plants infiltrated with Agrobacterium strain C58C1 harboring 35S:MKKK18-GFP and 35S:p19 constructs for transient expression. Four to five days after infiltration, the tobacco plants were treated with ABA and tissue samples were collected at the indicated time points. MKKK18 activity was assessed by the immunocomplex assay using anti-MKKK18 antibody and MBP as a substrate. MKKK18–GFP was detected with anti-GFP antibody. CBB staining of MBP confirmed equal loading. The above experiments were repeated several times with similar results.

    Article Snippet: The membranes were blocked for 1 h in PBS-T containing a 3% blocking solution, washed three times and incubated for 1 h with rabbit anti-GFP (1 : 200; sc-8334, Santa Cruz Biotechnology), StrepMAB-Classic (1 : 3,000; IBA BioTAGnology), anti-GST-Tag (1 : 5,000; Sigma), anti-MKKK18 antibody (AS13 2673, Agrisera), anti-ABI1 (AS12 1861, Agrisera) and anti-ABI2 (AS12 1871, Agrisera).

    Techniques: Activation Assay, Competitive Binding Assay, Recombinant, Incubation, Blocking Assay, Immunodetection, Immunoprecipitation, Activity Assay, Knock-Out, Isolation, Staining, Construct, Expressing

    Proteasome-dependent degradation of MKKK18 is ABA-dependent and regulated by ABI1. (A) qPCR analysis of MKKK18 transcript accumulation in response to treatment with 50 µM ABA for 90 min in WT Col-0, MKKK18oe , abi1td and abi1-2 strains. MKKK18 expression levels were determined using three biological replicates and were normalized against 18S rDNA. The results are displayed as mean log 2 fold change ± SE ( n = 9) of three independent experiments with consistent results. (B) MKKK18 stability in the cell-free degradation assay. GST–MKKK18 was incubated with 100 µg of protein extract from either WT Col-0 or abi1td protoplasts incubated with or without 100 µM MG132 for 6 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using anti-GST antibodies. Ponceau S staining confirmed equal loading. (C and D) Half-life plot for cell-free degradation of MKKK18 in WT Col-0 (C) and abi1td (D) extracts. Immunoblot images from each experiment were recorded simultaneously using a G:BOX Chemi XR5 fluorescence and chemiluminescence imaging system (Syngene), and the results were quantified using ImageJ software. (E) CHX treatment suppresses accumulation of MKKK18. Arabidopsis protoplasts expressing 35S:MKKK18-GFP were treated with 3 mM CHX, 3 mM CHX and 100 µM MG132, or given a mock treatment. The blot is representative of four experiments. MKKK18–GFP protein levels were determined by immunoblotting using anti-GFP antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control (mock) band (set as 1). (F) MKKK18 protein levels are modified by ABA. GST–MKKK18 was incubated with 100 µg of total protein extract isolated from WT Col-0 incubated with or without 50 µM ABA for 3 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using GST antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control band (set as 1).

    Journal: Plant and Cell Physiology

    Article Title: Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

    doi: 10.1093/pcp/pcv146

    Figure Lengend Snippet: Proteasome-dependent degradation of MKKK18 is ABA-dependent and regulated by ABI1. (A) qPCR analysis of MKKK18 transcript accumulation in response to treatment with 50 µM ABA for 90 min in WT Col-0, MKKK18oe , abi1td and abi1-2 strains. MKKK18 expression levels were determined using three biological replicates and were normalized against 18S rDNA. The results are displayed as mean log 2 fold change ± SE ( n = 9) of three independent experiments with consistent results. (B) MKKK18 stability in the cell-free degradation assay. GST–MKKK18 was incubated with 100 µg of protein extract from either WT Col-0 or abi1td protoplasts incubated with or without 100 µM MG132 for 6 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using anti-GST antibodies. Ponceau S staining confirmed equal loading. (C and D) Half-life plot for cell-free degradation of MKKK18 in WT Col-0 (C) and abi1td (D) extracts. Immunoblot images from each experiment were recorded simultaneously using a G:BOX Chemi XR5 fluorescence and chemiluminescence imaging system (Syngene), and the results were quantified using ImageJ software. (E) CHX treatment suppresses accumulation of MKKK18. Arabidopsis protoplasts expressing 35S:MKKK18-GFP were treated with 3 mM CHX, 3 mM CHX and 100 µM MG132, or given a mock treatment. The blot is representative of four experiments. MKKK18–GFP protein levels were determined by immunoblotting using anti-GFP antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control (mock) band (set as 1). (F) MKKK18 protein levels are modified by ABA. GST–MKKK18 was incubated with 100 µg of total protein extract isolated from WT Col-0 incubated with or without 50 µM ABA for 3 h in the dark. GST–MKKK18 protein levels at the indicated time points were determined by immunoblotting using GST antibodies. Ponceau S staining confirmed equal loading. MKKK18 protein bands were quantified using ImageJ software and normalized to the control band (set as 1).

    Article Snippet: The membranes were blocked for 1 h in PBS-T containing a 3% blocking solution, washed three times and incubated for 1 h with rabbit anti-GFP (1 : 200; sc-8334, Santa Cruz Biotechnology), StrepMAB-Classic (1 : 3,000; IBA BioTAGnology), anti-GST-Tag (1 : 5,000; Sigma), anti-MKKK18 antibody (AS13 2673, Agrisera), anti-ABI1 (AS12 1861, Agrisera) and anti-ABI2 (AS12 1871, Agrisera).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Degradation Assay, Incubation, Staining, Fluorescence, Imaging, Software, Modification, Isolation