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  • 94
    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
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    GE Healthcare glutathione sepharose 4b beads
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
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    GE Healthcare glutathione sepharose resin
    DDK promotes Sld3‐dependent Cdc45 recruitment Reaction scheme for Sld3/7/Cdc45 recruitment experiments. DNA substrate was bound to beads, and reaction mixtures were removed after each step. Immunoblots of recruitment reactions performed as described in (A), with the indicated proteins omitted. In (C), a mid‐reaction wash in high salt (0.5 M NaCl) buffer ( HSW ) was included following DDK phosphorylation as indicated. Recruitment reaction performed as in (A), using wild‐type (wt) or a Cdc45‐binding mutant (3E1) of Sld3. Schematic showing the position of Cdc45‐interacting region in Sld3. Alignments were generated in Jalview using Clustal. Sld3 from various fungal species is included (S.c., Saccharomyces cerevisiae , S.m., Saccharomyces mikatae, S.b., Saccharomyces bayanus, S.ca., Saccharomyces castellii, S.ku., Saccharomyces kudriavzevii ). Residue numbers correspond to S. cerevisiae Sld3. Residues were substituted for Glu as indicated. Wild‐type or mutant FLAG ‐Sld3 was added to an S‐phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot. Reaction scheme for in vitro replication reactions. Reaction mixtures were removed after each step. Reaction performed as in (G). Nascent DNA was separated in a 0.7% alkaline <t>agarose</t> gel in this and all subsequent replication reactions.
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    GE Healthcare glutathione sepharose cl4b beads
    DDK promotes Sld3‐dependent Cdc45 recruitment Reaction scheme for Sld3/7/Cdc45 recruitment experiments. DNA substrate was bound to beads, and reaction mixtures were removed after each step. Immunoblots of recruitment reactions performed as described in (A), with the indicated proteins omitted. In (C), a mid‐reaction wash in high salt (0.5 M NaCl) buffer ( HSW ) was included following DDK phosphorylation as indicated. Recruitment reaction performed as in (A), using wild‐type (wt) or a Cdc45‐binding mutant (3E1) of Sld3. Schematic showing the position of Cdc45‐interacting region in Sld3. Alignments were generated in Jalview using Clustal. Sld3 from various fungal species is included (S.c., Saccharomyces cerevisiae , S.m., Saccharomyces mikatae, S.b., Saccharomyces bayanus, S.ca., Saccharomyces castellii, S.ku., Saccharomyces kudriavzevii ). Residue numbers correspond to S. cerevisiae Sld3. Residues were substituted for Glu as indicated. Wild‐type or mutant FLAG ‐Sld3 was added to an S‐phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot. Reaction scheme for in vitro replication reactions. Reaction mixtures were removed after each step. Reaction performed as in (G). Nascent DNA was separated in a 0.7% alkaline <t>agarose</t> gel in this and all subsequent replication reactions.
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    GE Healthcare glutathione gsh sepharose beads
    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to <t>GSH-Sepharose</t> beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.
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    GE Healthcare glutathione sepharose 4b gs4b beads
    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to <t>GSH-Sepharose</t> beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.
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    GE Healthcare gamma glutathione sepharose beads
    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to <t>GSH-Sepharose</t> beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.
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    GE Healthcare glutathione sepharose 4ff beads
    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to <t>GSH-Sepharose</t> beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.
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    GE Healthcare slurry glutathione sepharose beads
    Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with <t>glutathione-Sepharose</t> beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.
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    GE Healthcare glutathione coated sepharose beads
    Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with <t>glutathione-Sepharose</t> beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.
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    GE Healthcare glutathione sepharose 4g beads
    Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with <t>glutathione-Sepharose</t> beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2018.02.011

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

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

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

    Interaction between HIV nucleocapsid (NC) protein and Tat ( A ) Plasmid expressing NC and plasmid expressing Tat were transformed to AH109 and the yeast was grown on media lacking histidine. Murine p53 and SV40 large T antigens were used as a positive control. HIV Vpu was used as a negative control. The lower panel shows yeast colonies resulting from interaction of the two proteins. Plasmids pGBK-NC, pGAD-Tat and pGAD-Vpu were constructed and utilized. pGBKT7-53 (murine p53) and pGADT7-T (SV40 large T antigen) were used as positive controls. Yeast strain AH109, transformed only with pGAD-Tat or pGAD-Vpu were used as negative controls; ( B ) Purified recombinant GST-fused NC was used to pulldown BKH21 cell lysate expressing Tat. GST was used as the negative control (third lane). pcDNA/V5-Tat was transfected to BHK-21 cells. After 24 h, a RIPA lysis buffer was added and lysis was performed at 4 °C for 30 min. The pellet was discarded after centrifugation and the lysate was obtained. GST or GST-NC and 30 μL of glutathione sepharose 4B bead was added to the lysate and the mixture was incubated at 4 °C overnight. HNGT buffer was used for washing and bound proteins were eluted after boiling with a 4XSDS sample buffer; ( C ) Flag-tagged NC and V5-tagged Tat or V5-tagged Vpu were expressed in HEK293 cells. The lysate was subjected to an immunoprecipitation with the anti-Flag antibody and detected with the anti-V5 antibody in a western blot analysis. The anti-IgG antibody was used a as the negative control; ( D ) Flag-tagged NC (green) and V5-tagged Tat (red) were expressed in HEK293 cells and observed under a confocal laser scanning microscope. At least three experiments were performed with essentially the same results.

    Journal: Viruses

    Article Title: Induced Degradation of Tat by Nucleocapsid (NC) via the Proteasome Pathway and Its Effect on HIV Transcription

    doi: 10.3390/v5041143

    Figure Lengend Snippet: Interaction between HIV nucleocapsid (NC) protein and Tat ( A ) Plasmid expressing NC and plasmid expressing Tat were transformed to AH109 and the yeast was grown on media lacking histidine. Murine p53 and SV40 large T antigens were used as a positive control. HIV Vpu was used as a negative control. The lower panel shows yeast colonies resulting from interaction of the two proteins. Plasmids pGBK-NC, pGAD-Tat and pGAD-Vpu were constructed and utilized. pGBKT7-53 (murine p53) and pGADT7-T (SV40 large T antigen) were used as positive controls. Yeast strain AH109, transformed only with pGAD-Tat or pGAD-Vpu were used as negative controls; ( B ) Purified recombinant GST-fused NC was used to pulldown BKH21 cell lysate expressing Tat. GST was used as the negative control (third lane). pcDNA/V5-Tat was transfected to BHK-21 cells. After 24 h, a RIPA lysis buffer was added and lysis was performed at 4 °C for 30 min. The pellet was discarded after centrifugation and the lysate was obtained. GST or GST-NC and 30 μL of glutathione sepharose 4B bead was added to the lysate and the mixture was incubated at 4 °C overnight. HNGT buffer was used for washing and bound proteins were eluted after boiling with a 4XSDS sample buffer; ( C ) Flag-tagged NC and V5-tagged Tat or V5-tagged Vpu were expressed in HEK293 cells. The lysate was subjected to an immunoprecipitation with the anti-Flag antibody and detected with the anti-V5 antibody in a western blot analysis. The anti-IgG antibody was used a as the negative control; ( D ) Flag-tagged NC (green) and V5-tagged Tat (red) were expressed in HEK293 cells and observed under a confocal laser scanning microscope. At least three experiments were performed with essentially the same results.

    Article Snippet: Exactly 4 μg each of GST and GST-NC and 30 μL of glutathione sepharose 4B bead (Amersham Biosciences) were added to the lysate and the mixture was incubated at 4 °C overnight.

    Techniques: Plasmid Preparation, Expressing, Transformation Assay, Positive Control, Negative Control, Construct, Purification, Recombinant, Transfection, Lysis, Centrifugation, Incubation, Immunoprecipitation, Western Blot, Laser-Scanning Microscopy

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

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms15022929

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

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

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0074773

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

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

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

    Epo1p binds to two different sites in Scs2p. (A, top) Domain structure of Scs2p. The major sperm domain (MSP) is separated from the C-terminal transmembrane segment (TMD) by a stretch of 93 residues. (bottom) Split-Ub interaction assay as in Fig. 1 B but between Epo1CRU and N ub -Scs2 1–225 and two N ub fusions that should not interact with Epo1CRU. (B) Eluates of Sepharose bead-coupled GST-Scs2 1–225 (lanes 1, 3, and 5) or GST (lanes 2, 4, and 6) incubated with yeast extracts containing GFP-tagged Epo1p (lanes 1 and 2) or bacterial extracts containing MBP-Epo1p (lanes 3 and 4) or MBP-Epo1 1–760 (lanes 5 and 6) were separated by SDS-PAGE. Western blots were probed with anti-GFP (lanes 1 and 2) or anti-MBP antibodies (lanes 3–6). Arrows indicate from top to bottom: MBP-Epo1p, Epo1-GFP, and MBP-Epo1 1–760 . Fragments of Epo1-GFP and MBP-Epo1p running

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: Epo1p binds to two different sites in Scs2p. (A, top) Domain structure of Scs2p. The major sperm domain (MSP) is separated from the C-terminal transmembrane segment (TMD) by a stretch of 93 residues. (bottom) Split-Ub interaction assay as in Fig. 1 B but between Epo1CRU and N ub -Scs2 1–225 and two N ub fusions that should not interact with Epo1CRU. (B) Eluates of Sepharose bead-coupled GST-Scs2 1–225 (lanes 1, 3, and 5) or GST (lanes 2, 4, and 6) incubated with yeast extracts containing GFP-tagged Epo1p (lanes 1 and 2) or bacterial extracts containing MBP-Epo1p (lanes 3 and 4) or MBP-Epo1 1–760 (lanes 5 and 6) were separated by SDS-PAGE. Western blots were probed with anti-GFP (lanes 1 and 2) or anti-MBP antibodies (lanes 3–6). Arrows indicate from top to bottom: MBP-Epo1p, Epo1-GFP, and MBP-Epo1 1–760 . Fragments of Epo1-GFP and MBP-Epo1p running

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Incubation, SDS Page, Western Blot

    The N-terminal 100 residues of Bem3p interact with the C-terminal coiled-coil regions of Epo1p. (A) Split-Ub assay as in Fig. 1 B but with cells coexpressing Epo1CRU and N ub fusions to Bem3p and its fragments. The N ub fusion to Guk1p should not interact and served as a control for the specificities of the observed interactions. (B) Cartoon indicating the positions of the N ub fragments and the domains of Bem3p. GAP, GTPase-activating domain. (C) As in Fig. 1 F , but with protein extracts of bacterial cells expressing his 6 -Epo1 852–943 (lanes 1, 3, and 4) or his 6 -Epo1 761–867 (lanes 2, 5, and 6) that were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 3 and 5) or GST-Bem3 1–100 (lanes 4 and 6). The vertical line indicates the removal of a lane loaded with a molecular mass marker. The inputs of GST and GST-Bem3 1–100 are shown as lanes 7 and 8 of a Coomassie-stained gel. Arrows indicate the running positions of GST-Bem3 1–100 and GST.

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: The N-terminal 100 residues of Bem3p interact with the C-terminal coiled-coil regions of Epo1p. (A) Split-Ub assay as in Fig. 1 B but with cells coexpressing Epo1CRU and N ub fusions to Bem3p and its fragments. The N ub fusion to Guk1p should not interact and served as a control for the specificities of the observed interactions. (B) Cartoon indicating the positions of the N ub fragments and the domains of Bem3p. GAP, GTPase-activating domain. (C) As in Fig. 1 F , but with protein extracts of bacterial cells expressing his 6 -Epo1 852–943 (lanes 1, 3, and 4) or his 6 -Epo1 761–867 (lanes 2, 5, and 6) that were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 3 and 5) or GST-Bem3 1–100 (lanes 4 and 6). The vertical line indicates the removal of a lane loaded with a molecular mass marker. The inputs of GST and GST-Bem3 1–100 are shown as lanes 7 and 8 of a Coomassie-stained gel. Arrows indicate the running positions of GST-Bem3 1–100 and GST.

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Expressing, Incubation, Marker, Staining

    Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different N ub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between N ub and C ub fusion. The identities of all Nub fusions are revealed in Table S2 . (B) Split-Ub interaction assay between Epo1CRU and selected N ub fusion proteins in WT, Δpea2 , Δspa2 , Δkel1 , and Δbem3 cells. Cells were grown to OD 600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. N ub without a C-terminally attached ORF (N ub −) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his 6 -Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1 761–943 (lanes 2 and 6), GST-Epo1 761–867 (lanes 3 and 7), or GST-Epo1 852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo1 1–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1 . (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his 6 -Spa2 1–535 -SNAP (lanes 1, 2, 7, and 8) or his 6 -Spa2 1–488 -SNAP (lanes 4, 5, 9, and 10) were first incubated with his 6 -Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1 761–943 . The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

    Journal: The Journal of Cell Biology

    Article Title: A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

    doi: 10.1083/jcb.201407126

    Figure Lengend Snippet: Epo1p interacts with members of the polarisome and Scs2p. (A) Split-Ub interaction assay of 48 yeast strains each coexpressing Epo1CRU with a different N ub fusion. Shown are quadruplets of each strain after 3 d of growth on medium containing 5-FOA. White boxes indicate the fusions that induce the growth of the strain reflecting the interaction between N ub and C ub fusion. The identities of all Nub fusions are revealed in Table S2 . (B) Split-Ub interaction assay between Epo1CRU and selected N ub fusion proteins in WT, Δpea2 , Δspa2 , Δkel1 , and Δbem3 cells. Cells were grown to OD 600 of 1 and 4 µl of this, and 10-fold serial dilutions were spotted on 5-FOA plates. N ub without a C-terminally attached ORF (N ub −) serves as a control for the specificity of the Split-Ub assays. (C) As in B, but selected interactions of Pea2CRU were compared between WT and Δepo1 cells. (D) Domain structure of Epo1p. Shown as blue rectangles are the three predicted coiled-coil (CC) regions. Numbers indicate amino acid positions of the putative start and end points of each domain. (E) As in A, but with fragments of Epo1p as CRU fusions and 16 independently generated diploids for each experiment shown after 4 d of growth. (F) Protein extracts of bacterial cells expressing his 6 -Pea2 (lanes 1–4) or yeast cells expressing MYC-tagged Kel1p (lanes 5–8) were incubated with glutathione-coupled Sepharose beads exposing bacterially expressed GST (lanes 1 and 5), GST-Epo1 761–943 (lanes 2 and 6), GST-Epo1 761–867 (lanes 3 and 7), or GST-Epo1 852–943 (lanes 4 and 8). Glutathione eluates were separated by SDS-PAGE and probed with anti-His (lanes 1–4) or anti-MYC (lanes 5–8) antibodies after Western blotting. (G) As in F, except bacterially expressed MBP-Epo1 (lanes 1 and 2) or MBP-Epo1 1–760 (lanes 3 and 4) were precipitated with bacterially expressed and Sepharose bead immobilized GST (lanes 2 and 4) or GST-Pea2p (lanes 1 and 3). The inputs for the experiments in F and G are shown in Fig. S1 . (H) Pea2p mediates the interaction between Epo1p and Spa2p. As in F, except bacterially expressed his 6 -Spa2 1–535 -SNAP (lanes 1, 2, 7, and 8) or his 6 -Spa2 1–488 -SNAP (lanes 4, 5, 9, and 10) were first incubated with his 6 -Pea2 (lanes 1 and 4) or left untreated (lanes 7 and 9) before being incubated with bacterially expressed and immobilized GST-Epo1 761–943 . The glutathione eluates are shown in lanes 1, 4, 7, and 9. The inputs for the experiment in lane 1 are shown in lanes 2 and 3. The inputs for the experiment in lane 4 are shown in lanes 5 and 6. The inputs for the experiment in lanes 7 and 9 are shown in lanes 8 and 10, respectively. The asterisk indicates a degradation product. Lanes 1–6 show cutouts of the same gel with the vertical line indicating the removal of an empty lane. (I) Architecture of the ER–cell tip tethering complex. Edges connecting nodes indicate direct (black) or potentially indirect (green) interactions. Blue rectangles indicate coiled-coil regions shown in D.

    Article Snippet: In vitro binding assay GST-tagged proteins were immobilized on glutathione-coupled Sepharose beads (GE Healthcare).

    Techniques: Generated, Expressing, Incubation, SDS Page, Western Blot

    GST pull-down validation of novel interactions identified by yeast mating assay. GST pull-down results for six pairs of interaction are shown as labeled. Purified GST-tagged Y. pestis T3SS proteins were immobilized onto the glutathione sepharose beads 4B and incubated overnight with the putative interacting proteins that were identified by yeast mating assay. After extensive washing, the beads were added with 2×SDS buffer and boiled, and the proteins were separated by SDS-PAGE electrophoresis. The separated proteins were transferred onto the PVDF membrane followed by immunoblotting using anti-GST or anti-His antibodies. The top panel shows the His-tagged proteins that were loaded in each pull-down assay for GST or GST-tagged Y. pestis protein. The middle and the lower panels show the pull-down proteins.

    Journal: PLoS ONE

    Article Title: Identification of Novel Protein-Protein Interactions of Yersinia pestis Type III Secretion System by Yeast Two Hybrid System

    doi: 10.1371/journal.pone.0054121

    Figure Lengend Snippet: GST pull-down validation of novel interactions identified by yeast mating assay. GST pull-down results for six pairs of interaction are shown as labeled. Purified GST-tagged Y. pestis T3SS proteins were immobilized onto the glutathione sepharose beads 4B and incubated overnight with the putative interacting proteins that were identified by yeast mating assay. After extensive washing, the beads were added with 2×SDS buffer and boiled, and the proteins were separated by SDS-PAGE electrophoresis. The separated proteins were transferred onto the PVDF membrane followed by immunoblotting using anti-GST or anti-His antibodies. The top panel shows the His-tagged proteins that were loaded in each pull-down assay for GST or GST-tagged Y. pestis protein. The middle and the lower panels show the pull-down proteins.

    Article Snippet: For purification of GST-tagged proteins, bacterial cells were resuspended in 1×PBS and lysed as above, and the proteins were purified with glutathione sepharose beads 4B (GE Healthcare) according to the standard protocol.

    Techniques: Labeling, Purification, Incubation, SDS Page, Electrophoresis, Pull Down Assay

    Identification of SelR and Clu interacting domains. (A–C) Co-IP analyses of the interacting domains of Clu which interacted with SelR. HEK293T cells were co-transfected with different plasmid pairs (pCMV-Myc- SelR′ and pcDNA3.1- Clu 290–314 -HA (A), pCMV-Myc- SelR′ and pcDNA3.1- Clu 315–381 -HA (B), pCMV-Myc- SelR′ and pcDNA3.1- Clu 382–460 -HA (C)). Cell lysates were analyzed by IP with HA-tag antibody and WB with Myc-tag antibody. 5% cell lysate (input) was added in a gel to be a positive control. (D) Pull-down analysis of the interaction between GST- Clu 315–381 and SelR. GST-Clu 315–381 was immobilized on glutathione Sepharose FF beads and its binding protein His-tagged SelR′ was analyzed by WB with His-tag antibody. GST was used as a negative control. (E–G) Co-IP analysis of SelR mutant and fragments interacting with Clu. HEK293T cells were co-transfected with plasmid pairs pcDNA3.1-HA- Clu and pCMV-Myc- SelR′′ (E) or pCMV-Myc-SelR1-94 (F), or pCMV-Myc-SelR19-82 (G). Cell lysates were analyzed by IP with HA-tag antibody and WB with Myc-tagged antibody. (H I) Pull-down analyses on the function of zinc ion in maintaining the interaction between SelR′-His and Clu 315–381 -HA. Clu 315–381 -HA was immobilized on the beads containing HA-tagged antibody. SelR′-His was then added to bind with Clu 315–381 . Metal chelating agents EDTA (H) and Selenocystine (I) were tested to remove the zinc ion bound to the tetrahedral of SelR′. (J) Model of the Clu and SelR interaction. The two Clu subunits are linked by five disulfide bonds. The shaded and hatched squares represent predicted disordered regions and predicted amphipathic helices, respectively. The central region of Clu 315–381 was shown to interact with the tetrahedral structure consisting of a zinc ion binding with four cysteine residues [Zn 2+ (Cys) 4 ] in SelR. Ab: Antibody; IP Ex: IP extract.

    Journal: PLoS ONE

    Article Title: Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer's Disease

    doi: 10.1371/journal.pone.0066384

    Figure Lengend Snippet: Identification of SelR and Clu interacting domains. (A–C) Co-IP analyses of the interacting domains of Clu which interacted with SelR. HEK293T cells were co-transfected with different plasmid pairs (pCMV-Myc- SelR′ and pcDNA3.1- Clu 290–314 -HA (A), pCMV-Myc- SelR′ and pcDNA3.1- Clu 315–381 -HA (B), pCMV-Myc- SelR′ and pcDNA3.1- Clu 382–460 -HA (C)). Cell lysates were analyzed by IP with HA-tag antibody and WB with Myc-tag antibody. 5% cell lysate (input) was added in a gel to be a positive control. (D) Pull-down analysis of the interaction between GST- Clu 315–381 and SelR. GST-Clu 315–381 was immobilized on glutathione Sepharose FF beads and its binding protein His-tagged SelR′ was analyzed by WB with His-tag antibody. GST was used as a negative control. (E–G) Co-IP analysis of SelR mutant and fragments interacting with Clu. HEK293T cells were co-transfected with plasmid pairs pcDNA3.1-HA- Clu and pCMV-Myc- SelR′′ (E) or pCMV-Myc-SelR1-94 (F), or pCMV-Myc-SelR19-82 (G). Cell lysates were analyzed by IP with HA-tag antibody and WB with Myc-tagged antibody. (H I) Pull-down analyses on the function of zinc ion in maintaining the interaction between SelR′-His and Clu 315–381 -HA. Clu 315–381 -HA was immobilized on the beads containing HA-tagged antibody. SelR′-His was then added to bind with Clu 315–381 . Metal chelating agents EDTA (H) and Selenocystine (I) were tested to remove the zinc ion bound to the tetrahedral of SelR′. (J) Model of the Clu and SelR interaction. The two Clu subunits are linked by five disulfide bonds. The shaded and hatched squares represent predicted disordered regions and predicted amphipathic helices, respectively. The central region of Clu 315–381 was shown to interact with the tetrahedral structure consisting of a zinc ion binding with four cysteine residues [Zn 2+ (Cys) 4 ] in SelR. Ab: Antibody; IP Ex: IP extract.

    Article Snippet: Pull-down Assay For the GST pull-down assay, a total of 40 µl glutathione Sepharose FF beads (GE Healthcare, Waukesha, WI, USA) were incubated with 50 µg GST-fused Clu315–381 , which was expressed in E. coli and purified by GST affinity chromatography for 1 h on ice in lysis buffer.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Positive Control, Binding Assay, Negative Control, Mutagenesis

    DDK promotes Sld3‐dependent Cdc45 recruitment Reaction scheme for Sld3/7/Cdc45 recruitment experiments. DNA substrate was bound to beads, and reaction mixtures were removed after each step. Immunoblots of recruitment reactions performed as described in (A), with the indicated proteins omitted. In (C), a mid‐reaction wash in high salt (0.5 M NaCl) buffer ( HSW ) was included following DDK phosphorylation as indicated. Recruitment reaction performed as in (A), using wild‐type (wt) or a Cdc45‐binding mutant (3E1) of Sld3. Schematic showing the position of Cdc45‐interacting region in Sld3. Alignments were generated in Jalview using Clustal. Sld3 from various fungal species is included (S.c., Saccharomyces cerevisiae , S.m., Saccharomyces mikatae, S.b., Saccharomyces bayanus, S.ca., Saccharomyces castellii, S.ku., Saccharomyces kudriavzevii ). Residue numbers correspond to S. cerevisiae Sld3. Residues were substituted for Glu as indicated. Wild‐type or mutant FLAG ‐Sld3 was added to an S‐phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot. Reaction scheme for in vitro replication reactions. Reaction mixtures were removed after each step. Reaction performed as in (G). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.

    Journal: The EMBO Journal

    Article Title: Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

    doi: 10.15252/embj.201593552

    Figure Lengend Snippet: DDK promotes Sld3‐dependent Cdc45 recruitment Reaction scheme for Sld3/7/Cdc45 recruitment experiments. DNA substrate was bound to beads, and reaction mixtures were removed after each step. Immunoblots of recruitment reactions performed as described in (A), with the indicated proteins omitted. In (C), a mid‐reaction wash in high salt (0.5 M NaCl) buffer ( HSW ) was included following DDK phosphorylation as indicated. Recruitment reaction performed as in (A), using wild‐type (wt) or a Cdc45‐binding mutant (3E1) of Sld3. Schematic showing the position of Cdc45‐interacting region in Sld3. Alignments were generated in Jalview using Clustal. Sld3 from various fungal species is included (S.c., Saccharomyces cerevisiae , S.m., Saccharomyces mikatae, S.b., Saccharomyces bayanus, S.ca., Saccharomyces castellii, S.ku., Saccharomyces kudriavzevii ). Residue numbers correspond to S. cerevisiae Sld3. Residues were substituted for Glu as indicated. Wild‐type or mutant FLAG ‐Sld3 was added to an S‐phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot. Reaction scheme for in vitro replication reactions. Reaction mixtures were removed after each step. Reaction performed as in (G). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.

    Article Snippet: The supernatant was subjected to GST affinity purification by incubation with 0.8 ml packed bead volume of glutathione sepharose resin (GE Healthcare) that had been pre‐washed in buffer A/500 mM KCl.

    Techniques: Western Blot, Binding Assay, Mutagenesis, Generated, Immunoprecipitation, In Vitro, Agarose Gel Electrophoresis

    Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to GSH-Sepharose beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.

    Journal: Molecular Biology of the Cell

    Article Title: Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase

    doi:

    Figure Lengend Snippet: Mpr1 functions upstream of the Mcs4 response regulator. (A) Oxidative stress induces physical association between Mpr1 and Mcs4. Strain CA337 has chromosomal mcs4 + tagged with the sequence encoding the myc epitope. This strain was transformed with pREP1-KZ-mpr1 and pREP1-KZ-mpr1HQ plasmids, which express GST fusion proteins of wild-type and His-221→Gln mutant Mpr1, respectively, under the regulation of the thiamine-repressible nmt1 promoter. The transformants were grown in EMM2 medium with 0.03 μM thiamine to induce expression of the GST fusion proteins at a low level and treated with either oxidative stress induced by 0.3 mM H 2 O 2 (left panels) or high-osmolarity stress induced by 0.6 M KCl (right panels) for the indicated times. Cell lysates were absorbed to GSH-Sepharose beads, and after extensive washes, proteins bound to the beads (GSH-Beads) were analyzed by immunoblotting with anti-myc and anti-GST antibodies. The amount of Mcs4 detected in the crude cell lysates (Lysate) did not change significantly after the stress treatments. (B) Wild-type (KS1376), Δmcs4 (CA220), Δmpr1 (CA279), and Δmcs4 Δmpr1 (CA420) strains carrying the spc1:HA6H allele were grown to midlog phase at 30°C in YES medium and treated with oxidative stress induced by 0.3 mM H 2 O 2 . Aliquots of cells were harvested at the indicated times, and Spc1 was purified by Ni-NTA chromatography, followed by immunoblotting with anti-phospho-p38 and anti-HA antibodies. The pattern of Spc1 activation in the Δmcs4 Δmpr1 double mutant is identical to that in the Δmcs4 mutant before and after oxidative stress. (C) Wild-type (KS1376), Δmcs4 (CA220), and Δmpr1 (CA279) strains were treated with high-osmolarity stress induced by 0.6 M KCl, and Spc1 activation was examined as described for B.

    Article Snippet: The protein concentration of each lysate was normalized with the use of a Bio-Rad (Richmond, CA) protein assay before immunoblot analysis of Mcs4 (see Figure A, bottom panel) and incubation with 10 μl (bed volume) of glutathione (GSH)-Sepharose beads (Amersham Pharmacia).

    Techniques: Sequencing, Transformation Assay, Mutagenesis, Expressing, Purification, Chromatography, Activation Assay

    Effects of the interaction between GlpK and the KPN00353 variants on 1,3-PD production. After the interaction of GST-tagged GlpK with wild-type or variant His-tagged KPN00353, the proteins captured on GSH-Sepharose beads were analyzed by Western blotting using either anti-GST antibody (α-GST) or anti-His antibody (α-His). From the Western blotting results, we defined the binding affinity of GlpK and wild-type KPN00353 (WT) to be 1+. The binding affinity of GlpK and the KPN00353 variant was comparable to that of GlpK and H65wt. H65wt, E. coli with pET30b::353WT; H65D, E. coli with pET30b::H65D; H65E, E. coli with pET30b::H65E; H65R, E. coli with pET30b::H65R; H65Q, E. coli with pET30b::H65Q; H110Q, E. coli with pET30b::H110Q. The 1,3-PD production of K. pneumoniae with pBAD33 (Vec) after incubation for 4 h was 0.186 g/L as 100%. The 1,3-PD production of K. pneumoniae with plasmids expressing wild-type or variant KPN00353 was compared to that of Vec. The residual glycerol in the medium was compared to the glycerol concentration in fresh MCM. H65wt, K. pneumoniae with pBAD33::Histaq353WT; H65D, K. pneumoniae with pBAD33::Histaq353H65D; H65E, K. pneumoniae with pBAD33::Histaq353H65E; H65R, K. pneumoniae with pBAD33::Histaq353H65R; H65Q, K. pneumoniae with pBAD33::Histaq353H65Q; H110Q, K. pneumoniae with pBAD33::Histaq353H110Q.

    Journal: Frontiers in Microbiology

    Article Title: The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae

    doi: 10.3389/fmicb.2017.02441

    Figure Lengend Snippet: Effects of the interaction between GlpK and the KPN00353 variants on 1,3-PD production. After the interaction of GST-tagged GlpK with wild-type or variant His-tagged KPN00353, the proteins captured on GSH-Sepharose beads were analyzed by Western blotting using either anti-GST antibody (α-GST) or anti-His antibody (α-His). From the Western blotting results, we defined the binding affinity of GlpK and wild-type KPN00353 (WT) to be 1+. The binding affinity of GlpK and the KPN00353 variant was comparable to that of GlpK and H65wt. H65wt, E. coli with pET30b::353WT; H65D, E. coli with pET30b::H65D; H65E, E. coli with pET30b::H65E; H65R, E. coli with pET30b::H65R; H65Q, E. coli with pET30b::H65Q; H110Q, E. coli with pET30b::H110Q. The 1,3-PD production of K. pneumoniae with pBAD33 (Vec) after incubation for 4 h was 0.186 g/L as 100%. The 1,3-PD production of K. pneumoniae with plasmids expressing wild-type or variant KPN00353 was compared to that of Vec. The residual glycerol in the medium was compared to the glycerol concentration in fresh MCM. H65wt, K. pneumoniae with pBAD33::Histaq353WT; H65D, K. pneumoniae with pBAD33::Histaq353H65D; H65E, K. pneumoniae with pBAD33::Histaq353H65E; H65R, K. pneumoniae with pBAD33::Histaq353H65R; H65Q, K. pneumoniae with pBAD33::Histaq353H65Q; H110Q, K. pneumoniae with pBAD33::Histaq353H110Q.

    Article Snippet: Glutathione-sepharose (GSH) beads (50 μL; GE Healthcare, United Kingdom) were then added to the spent supernatant, and the mixture was incubated with mild shaking for 1 h at 4°C.

    Techniques: Variant Assay, Western Blot, Binding Assay, Incubation, Expressing, Concentration Assay

    Interaction between KPN00353 and GlpK. GST-tagged GlpK (GST-GlpK) was allowed to interact with His-tagged protein (lane 2, 3 in A,B ), and then the proteins captured on GSH-Sepharose beads were analyzed by (i) SDS-PAGE followed by Coomassie Blue staining and (ii) Western blotting using anti-His antibody. The purified proteins were analyzed simultaneously with the control (lane 4, 5, 6 in A,B ). The protein marker is shown in lane 1. His-353, His-tagged KPN00353; His-MrkD, His-tagged MrkD; GST, glutathione S-transferase.

    Journal: Frontiers in Microbiology

    Article Title: The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae

    doi: 10.3389/fmicb.2017.02441

    Figure Lengend Snippet: Interaction between KPN00353 and GlpK. GST-tagged GlpK (GST-GlpK) was allowed to interact with His-tagged protein (lane 2, 3 in A,B ), and then the proteins captured on GSH-Sepharose beads were analyzed by (i) SDS-PAGE followed by Coomassie Blue staining and (ii) Western blotting using anti-His antibody. The purified proteins were analyzed simultaneously with the control (lane 4, 5, 6 in A,B ). The protein marker is shown in lane 1. His-353, His-tagged KPN00353; His-MrkD, His-tagged MrkD; GST, glutathione S-transferase.

    Article Snippet: Glutathione-sepharose (GSH) beads (50 μL; GE Healthcare, United Kingdom) were then added to the spent supernatant, and the mixture was incubated with mild shaking for 1 h at 4°C.

    Techniques: SDS Page, Staining, Western Blot, Purification, Marker

    Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with glutathione-Sepharose beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.

    Journal: Cellular signalling

    Article Title: G?o/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

    doi: 10.1016/j.cellsig.2008.02.008

    Figure Lengend Snippet: Direct interaction between Gα o and RGS20. (A) Pull-down of RGS20 by GST-Gα o -WT (wild-type) and GST-Gα o -Q/L (constitutively active mutant). COS-7 cells grown in 100 mm dishes were transfected with the pBK-FLAG-RGS20 construct. The extracted proteins were incubated with glutathione-Sepharose beads conjugated to GST protein alone or to the fusion proteins GST-Gα o -WTor GST-Gα o -Q/L. The proteins eluted out of the collected beads were separated by SDS-PAGE (12% acrylamide), and RGS20 was visualized by immunoblotting with M2-FLAG antibody. (B) The cell lysates containing the FLAG-RGS20 used as inputs for the GST pull down were probed with M2-FLAG antibodies. A non-specific band migrating below the 30 kDa marker is seen with the lot of M2-FLAG antibody used. (C) Anti-FLAG western blot in COS-7 cells transfected or not with FLAG-RGS20. COS-7 cells were transfected with the pBK-FLAG-RGS20 construct or the vector alone (pBK-FLAG). 20 μg of the extracted proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose paper and probed for the FLAG-tag with a M2-FLAG monoclonal antibody. (D) Pull-down of Gα o by GST-RGS20. COS-7 cells were transfected with different amounts (1, 0.5 or 0.1 μg) of expression constructs for Gα o -WTor Gα o -Q/L mutant. The extracted proteins were incubated with equal amounts of glutathione-sepharose beads conjugated to the fusion protein GST-RGS20, and the pulled-down Gα o was revealed by SDS-PAGE/WB with an anti-Gα o antibody. (E) Approximately 5% of the lysates used as pull-down input was probed by SDS-PAGE/WB for Gα o protein.

    Article Snippet: Soluble GST-fused proteins were purified from crude bacterial lysates by incubation at 4 °C for 60 min with 50% slurry glutathione-sepharose beads (Glutathione-Sepharose 4B, Amersham).

    Techniques: Mutagenesis, Transfection, Construct, Incubation, SDS Page, Marker, Western Blot, Plasmid Preparation, FLAG-tag, Expressing

    Gα o stimulates the ubiquitination of RGS20. (A) COS-7 cells were transfected with pBK-FLAG-RGS20 with and without pcDNA3.1-Gα o -Q/L and, after 24 h, treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. The harvested cell pellets were lysed in denaturing conditions at 95 °C in 2% SDS to suppress deubiquitinating and proteolytic activities. RGS20 was immunoprecipitated from 0.5–1 mg of extracted proteins with 4 μg of M2-FLAG antibody and 30 μl of protein-G-agarose. Immunoprecipitated proteins were separated by SDS-PAGE with 4–12% gradient gel and ubiquitinated RGS20 was detected by Western blot with a monoclonal antibody anti-ubiquitin. (B) COS-7 cells were transfected with pBK-FLAG-RGS20 with and without pcDNA3.1-Gα o -Q/L and, after 24 h, treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. Cell lysis and immunoprecipitation of RGS20 were carried out as in Panel A. Immunoprecipitated proteins were separated by SDS-PAGE with 4–12% gradient gel and RGS20 was detected by Western blot with M2-FLAG antibody. (C) COS-7 cells were transfected with either pBK-FLAG-RGS20 or pcDNA3.1-FLAG-Traf2, in the presence or absence of pcDNA3.1-Gα o -Q/L, and all in the presence of pcDNA3.1-HA-Ubiquitin. After 24 h, cells were treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. The harvested cells were lysed in denaturing conditions at 95 °C in 2% SDS. RGS20 or Traf2 were immunoprecipitated from 0.5–1 mg of extracted proteins with 4 μg of M2-FLAG antibody and 30 μl of protein-G-agarose. Immunoprecipitated proteins were separated by SDS-PAGE in a 4–12% gradient gel and ubiquitinated species were detected by Western blot with anti-HA antibody (for HA-ubiquitin). (D) Corresponding whole cell lysates from C, probed for Gα o (top panel) and FLAG (bottom panel).

    Journal: Cellular signalling

    Article Title: G?o/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

    doi: 10.1016/j.cellsig.2008.02.008

    Figure Lengend Snippet: Gα o stimulates the ubiquitination of RGS20. (A) COS-7 cells were transfected with pBK-FLAG-RGS20 with and without pcDNA3.1-Gα o -Q/L and, after 24 h, treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. The harvested cell pellets were lysed in denaturing conditions at 95 °C in 2% SDS to suppress deubiquitinating and proteolytic activities. RGS20 was immunoprecipitated from 0.5–1 mg of extracted proteins with 4 μg of M2-FLAG antibody and 30 μl of protein-G-agarose. Immunoprecipitated proteins were separated by SDS-PAGE with 4–12% gradient gel and ubiquitinated RGS20 was detected by Western blot with a monoclonal antibody anti-ubiquitin. (B) COS-7 cells were transfected with pBK-FLAG-RGS20 with and without pcDNA3.1-Gα o -Q/L and, after 24 h, treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. Cell lysis and immunoprecipitation of RGS20 were carried out as in Panel A. Immunoprecipitated proteins were separated by SDS-PAGE with 4–12% gradient gel and RGS20 was detected by Western blot with M2-FLAG antibody. (C) COS-7 cells were transfected with either pBK-FLAG-RGS20 or pcDNA3.1-FLAG-Traf2, in the presence or absence of pcDNA3.1-Gα o -Q/L, and all in the presence of pcDNA3.1-HA-Ubiquitin. After 24 h, cells were treated with 10 μM MG132 or vehicle (0.1% DMSO) for 10 h. The harvested cells were lysed in denaturing conditions at 95 °C in 2% SDS. RGS20 or Traf2 were immunoprecipitated from 0.5–1 mg of extracted proteins with 4 μg of M2-FLAG antibody and 30 μl of protein-G-agarose. Immunoprecipitated proteins were separated by SDS-PAGE in a 4–12% gradient gel and ubiquitinated species were detected by Western blot with anti-HA antibody (for HA-ubiquitin). (D) Corresponding whole cell lysates from C, probed for Gα o (top panel) and FLAG (bottom panel).

    Article Snippet: Soluble GST-fused proteins were purified from crude bacterial lysates by incubation at 4 °C for 60 min with 50% slurry glutathione-sepharose beads (Glutathione-Sepharose 4B, Amersham).

    Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Lysis