glutathione sepharose  (GE Healthcare)

 
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    Name:
    Glutathione sepharose 4B
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    GS2376858
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    Structured Review

    GE Healthcare glutathione sepharose
    ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione <t>sepharose</t> beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

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    Images

    1) Product Images from "Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo"

    Article Title: Interaction between the cellular E3 ubiquitin ligase SIAH-1 and the viral immediate-early protein ICP0 enables efficient replication of Herpes Simplex Virus type 2 in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201880

    ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.
    Figure Legend Snippet: ICP0 interacts with SIAH-1 via two minimal interaction motifs. ( A ) Schematic representation of HSV-2 ICP0 indicating the position of the RING domain (yellow), the USP7 interaction domain (blue), the nuclear localization signal (brown) and the two SIAH interaction motifs VxP1 and VxP2 (red). The primary amino acid sequence surrounding the predicted SIAH binding motifs is depicted together with the consensus motif and the position of the inactivating NxN mutation. ( B ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and the cell lysates were incubated with GST or GST-SIAH-1-loaded glutathione sepharose beads. The upper SDS-PAGE gel shows a Coomassie staining of the respective input control (lysate) and the eluates from the GST or GST-SIAH-1 beads. Below, a contrast enhanced section of the gel with putative ICP0 bands indicated by asterisks. ICP0 was detected by Western blotting using an antibody against the C-terminal GFP tag. Size markers in kDa. ( C ) HEK293T cells were transfected with plasmids encoding GFP-tagged ICP0 and its mutants and HA-tagged SIAH-1. The ICP0-GFP proteins were immunoprecipitated from the lysates, ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against the GFP and HA-tags. ( D ) HEK293T cells were transfected with plasmids encoding the inactive mutant HA-SIAH-1 C44S and GFP-ICP0 ΔRING or GFP-ICP0 ΔRING/NxN1/2 as indicated. Immunoprecipitation from the cell lysates was performed using control mouse IgG or anti-SIAH-1. ICP0 and SIAH-1 were detected by SDS-PAGE and Western blotting using antibodies against SIAH-1 or the GFP-tag. The lower panel shows the analysis of the RIPA buffer-insoluble pellet after cell lysis.

    Techniques Used: Sequencing, Binding Assay, Mutagenesis, Transfection, Incubation, SDS Page, Staining, Western Blot, Hemagglutination Assay, Immunoprecipitation, Lysis

    Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.
    Figure Legend Snippet: Virally expressed ICP0 NxN1/2 does not bind to SIAH-1. ( A ) U2OS cells were infected for 48 h with the indicated mutants and HSV-2 strain MS (MS wt) at an MOI 0.01 pfu/cell. Cell lysates were incubated with GST-SIAH-1-loaded glutathione sepharose beads. Eluates were analyzed by SDS-PAGE and Western blotting using antibodies directed against HSV-2 ICP0 and GFP. ( B ) Input controls (10%) of the GST-pulldown were analyzed as before using ICP0-specific antibody.

    Techniques Used: Infection, Mass Spectrometry, Incubation, SDS Page, Western Blot

    2) Product Images from "The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase"

    Article Title: The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase

    Journal:

    doi: 10.1016/j.cell.2014.03.046

    TRPM7 cleavage fragments identified in multiple cell lines and tissues A. TRPM7 protein cleavage fragments in mouse mesangial SV40 mes13 cells. Cells were extracted with TBS/1% NP40. Endogenous TRPM7 was immunoprecipitated (IP’d) from lysates with TRPM7 C-terminal mouse monoclonal antibody (αA25) or normal mouse IgG and probed on WB with anti-C-terminal rabbit antibody (αC47). C-terminally HA-tagged TRPM7 (expressed=expr) was IP’d with anti-HA-agarose (αHA) from SV40 mes13 cells stably expressing recombinant protein and probed on WB with αHA-peroxidase conjugate. Scale ( left ) indicates the molecular weight of major bands calculated from their electrophoretic mobility relative to standard molecular weight markers. Cartoon ( right ) shows the approximate position of cleavage sites; K indicates kinase domain. B. TRPM7 cleavage pattern in 8 distinct cell lines. Mouse mesangial (SV40 mes13), macrophage (RAW 264.7), mESC, human B-lymphocyte (Raji), Caco-2 (colon epithelial), prostate (metastatic LNCaP and non-metastatic RWPE1), and embryonic kidney (HEK-293) cells were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from each tissue. Information about the relative content of the full length TRPM7 and the cleaved fragments is contained in each individual lane, which are intentionally not normalized to control protein. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown). mESCs were generated as described in Experimental Procedures from WT or TrpM7−/− (KO) blastocysts. The lower panel in the mESC column shows equal actin content in both mESC lysates. Samples run on different gels are combined in the figure and aligned against identical molecular weight markers. C. TRPM7 cleavage pattern in different mouse tissues. Freshly isolated mouse organs were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from in each tissue. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown).
    Figure Legend Snippet: TRPM7 cleavage fragments identified in multiple cell lines and tissues A. TRPM7 protein cleavage fragments in mouse mesangial SV40 mes13 cells. Cells were extracted with TBS/1% NP40. Endogenous TRPM7 was immunoprecipitated (IP’d) from lysates with TRPM7 C-terminal mouse monoclonal antibody (αA25) or normal mouse IgG and probed on WB with anti-C-terminal rabbit antibody (αC47). C-terminally HA-tagged TRPM7 (expressed=expr) was IP’d with anti-HA-agarose (αHA) from SV40 mes13 cells stably expressing recombinant protein and probed on WB with αHA-peroxidase conjugate. Scale ( left ) indicates the molecular weight of major bands calculated from their electrophoretic mobility relative to standard molecular weight markers. Cartoon ( right ) shows the approximate position of cleavage sites; K indicates kinase domain. B. TRPM7 cleavage pattern in 8 distinct cell lines. Mouse mesangial (SV40 mes13), macrophage (RAW 264.7), mESC, human B-lymphocyte (Raji), Caco-2 (colon epithelial), prostate (metastatic LNCaP and non-metastatic RWPE1), and embryonic kidney (HEK-293) cells were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from each tissue. Information about the relative content of the full length TRPM7 and the cleaved fragments is contained in each individual lane, which are intentionally not normalized to control protein. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown). mESCs were generated as described in Experimental Procedures from WT or TrpM7−/− (KO) blastocysts. The lower panel in the mESC column shows equal actin content in both mESC lysates. Samples run on different gels are combined in the figure and aligned against identical molecular weight markers. C. TRPM7 cleavage pattern in different mouse tissues. Freshly isolated mouse organs were extracted and IP’d as described in A . Extracts demonstrate the relative amounts of cleaved TRPM7 isolated from in each tissue. No positive bands were found from the same tissue extracts IP’d with normal mouse IgG (not shown).

    Techniques Used: Immunoprecipitation, Western Blot, Hemagglutination Assay, Stable Transfection, Expressing, Recombinant, Molecular Weight, Isolation, Generated, Gene Knockout

    3) Product Images from "Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism"

    Article Title: Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

    Journal:

    doi: 10.1128/MCB.01203-13

    Direct interaction between the cytosolic domain of VAMP2 and the SNARE motif of syntaxin4. (A) A 10-μg amount of VAMP2-PrA or PrA bound to IgG-Sepharose (10-μl bed volume) was incubated with a 10× molar excess of the cytosolic
    Figure Legend Snippet: Direct interaction between the cytosolic domain of VAMP2 and the SNARE motif of syntaxin4. (A) A 10-μg amount of VAMP2-PrA or PrA bound to IgG-Sepharose (10-μl bed volume) was incubated with a 10× molar excess of the cytosolic

    Techniques Used: Incubation

    Effect of Munc18c on syntaxin4/SNAP23/VAMP2 SNARE complex formation. A 30- to 50-μg amount of Sx4-GST bound to glutathione-Sepharose (10-μl bed volume) was preincubated for 2 h at 4°C in a 1-ml volume with a 10× molar excess
    Figure Legend Snippet: Effect of Munc18c on syntaxin4/SNAP23/VAMP2 SNARE complex formation. A 30- to 50-μg amount of Sx4-GST bound to glutathione-Sepharose (10-μl bed volume) was preincubated for 2 h at 4°C in a 1-ml volume with a 10× molar excess

    Techniques Used:

    4) Product Images from "The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways"

    Article Title: The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200705021

    Identification of motifs required for direct EspF and SNX9 interactions. (A) Glutathione-Sepharose pull-down with 10 μg of GST-EspF (residues 48–206) mixed with the [ 35 S]-methionine proteins indicated (left diagram). Autoradiograph of GST pulldown (left) and of 1/20th input of 35 S-labeled NCK1, NCK2, SNX9 (residues 1–111), and GRB2 is shown (right). (B) HEK293A cells were cotransfected with EGFP-EspF and V5-tagged proteins indicated (left diagram). Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (left). Cell lysates were probed by V5 or GFP immunoblot to show input levels. (C) Logos plot of the SNX9 binding consensus sequence derived by phage display experiments (left). Alignment of 13 unique SNX9 binding sequences used to derive the consensus is shown. The invariant arginine (blue) and highly conserved residues (gray) are highlighted. (D) Peptide array analysis of the SNX9-binding sites on EspF. Top: diagram of EspF residues 1–166 used for the peptide scanning experiments. Middle: ultraviolet (UV) illumination shows the qualitative amount of each peptide synthesized (top). Bottom: solid-phase binding of 35 S-SNX9 to 15-mer EspF peptides was assessed by autoradiography. An alignment of EspF-binding peptides from two SNX9 binding series is shown. (E) Saturation binding curves were generated with increasing concentrations of GST-SNX9-SH3 (left) to a fixed concentration of EspF peptide by fluorescence polarization (see Materials and methods). (F) HEK293A cells were cotransfected with EGFP-EspF or triple mutant EGFP-EspF-D3 (top diagram) and V5-tagged SNX9. Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (top panel). Cell lysates were probed by V5 or GFP immunoblot to show input levels (bottom two panels).
    Figure Legend Snippet: Identification of motifs required for direct EspF and SNX9 interactions. (A) Glutathione-Sepharose pull-down with 10 μg of GST-EspF (residues 48–206) mixed with the [ 35 S]-methionine proteins indicated (left diagram). Autoradiograph of GST pulldown (left) and of 1/20th input of 35 S-labeled NCK1, NCK2, SNX9 (residues 1–111), and GRB2 is shown (right). (B) HEK293A cells were cotransfected with EGFP-EspF and V5-tagged proteins indicated (left diagram). Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (left). Cell lysates were probed by V5 or GFP immunoblot to show input levels. (C) Logos plot of the SNX9 binding consensus sequence derived by phage display experiments (left). Alignment of 13 unique SNX9 binding sequences used to derive the consensus is shown. The invariant arginine (blue) and highly conserved residues (gray) are highlighted. (D) Peptide array analysis of the SNX9-binding sites on EspF. Top: diagram of EspF residues 1–166 used for the peptide scanning experiments. Middle: ultraviolet (UV) illumination shows the qualitative amount of each peptide synthesized (top). Bottom: solid-phase binding of 35 S-SNX9 to 15-mer EspF peptides was assessed by autoradiography. An alignment of EspF-binding peptides from two SNX9 binding series is shown. (E) Saturation binding curves were generated with increasing concentrations of GST-SNX9-SH3 (left) to a fixed concentration of EspF peptide by fluorescence polarization (see Materials and methods). (F) HEK293A cells were cotransfected with EGFP-EspF or triple mutant EGFP-EspF-D3 (top diagram) and V5-tagged SNX9. Anti-GFP immunoprecipitations (IP) were probed by V5 immunoblot (IB) (top panel). Cell lysates were probed by V5 or GFP immunoblot to show input levels (bottom two panels).

    Techniques Used: Autoradiography, Labeling, Binding Assay, Sequencing, Derivative Assay, Peptide Microarray, Synthesized, Generated, Concentration Assay, Fluorescence, Mutagenesis

    SNX9 is the major binding partner for EspF in polarized epithelial cells. (A) TER measurements of polarized T84 colonic epithelial cells infected with EPEC and the indicated EspF mutants. The average change in TER in three independent experiments is shown. (B and C) Fluorescence microscopy of MDCK cells uninfected (B) or infected with EPEC for 4 h (C). Tight junction morphology was detected by anti-occludin immunocytochemistry. (D) Schematic of TAP tagged EspF is shown. Western blot of cellular lysates collected from stable MDCK cells expressing TAP-flag-EspF or mutant TAP-flag-EspF-D3. Anti-flag was used to probe EspF expression (top blot) and actin was used as a protein loading control (bottom blot). (E and F) Fluorescence microscopy of MDCK cells expressing TAP-EspF and TAP-EspF-D3. Tight junction morphology was detected by anti-occludin immunocytochemistry. (G) MDCK parental or Tap-EspF cellular lysates were incubated with anti-flag agarose and the resulting immuno-complexes were subjected to SDS-PAGE and stained with Coomassie. Proteins identified by mass spectrometry are indicated. “NS” designates nonspecific interacting proteins and “IgG LC” is the immunoglobulin light chain. (H) Anti-SNX9 immunofluorescence microscopy of CaCo 2 cells infected with the indicated EPEC strains for 3 h. Boxed area is a 4× magnification of the area indicated. Cellular borders are outlined. Bar = 15 μm. (I) Immunoblot of EspF from wild-type EPEC (Wt) or EPEC espF − strains carrying plasmids encoding wild-type EspF ( pespf ) or mutant EspF-D3 ( pespf-D3 ) tagged with the myc epitope. Type III secreted EspF harvested from media supernatants (top) and from whole bacterial lysates (bottom) are shown. Concentrations of IPTG are indicated below.
    Figure Legend Snippet: SNX9 is the major binding partner for EspF in polarized epithelial cells. (A) TER measurements of polarized T84 colonic epithelial cells infected with EPEC and the indicated EspF mutants. The average change in TER in three independent experiments is shown. (B and C) Fluorescence microscopy of MDCK cells uninfected (B) or infected with EPEC for 4 h (C). Tight junction morphology was detected by anti-occludin immunocytochemistry. (D) Schematic of TAP tagged EspF is shown. Western blot of cellular lysates collected from stable MDCK cells expressing TAP-flag-EspF or mutant TAP-flag-EspF-D3. Anti-flag was used to probe EspF expression (top blot) and actin was used as a protein loading control (bottom blot). (E and F) Fluorescence microscopy of MDCK cells expressing TAP-EspF and TAP-EspF-D3. Tight junction morphology was detected by anti-occludin immunocytochemistry. (G) MDCK parental or Tap-EspF cellular lysates were incubated with anti-flag agarose and the resulting immuno-complexes were subjected to SDS-PAGE and stained with Coomassie. Proteins identified by mass spectrometry are indicated. “NS” designates nonspecific interacting proteins and “IgG LC” is the immunoglobulin light chain. (H) Anti-SNX9 immunofluorescence microscopy of CaCo 2 cells infected with the indicated EPEC strains for 3 h. Boxed area is a 4× magnification of the area indicated. Cellular borders are outlined. Bar = 15 μm. (I) Immunoblot of EspF from wild-type EPEC (Wt) or EPEC espF − strains carrying plasmids encoding wild-type EspF ( pespf ) or mutant EspF-D3 ( pespf-D3 ) tagged with the myc epitope. Type III secreted EspF harvested from media supernatants (top) and from whole bacterial lysates (bottom) are shown. Concentrations of IPTG are indicated below.

    Techniques Used: Binding Assay, Infection, Fluorescence, Microscopy, Immunocytochemistry, Western Blot, Expressing, Mutagenesis, Incubation, SDS Page, Staining, Mass Spectrometry, Liquid Chromatography, Immunofluorescence

    5) Product Images from "Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE"

    Article Title: Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

    Journal:

    doi: 10.1128/JB.187.1.249-256.2005

    Removal of GST-containing oligomers. EpsE was treated with glutathione Sepharose and applied to a Superose 6 column. Fractions of 0.5 ml were collected and analyzed by SDS-PAGE and silver staining (lower panel). These fractions were compared with the
    Figure Legend Snippet: Removal of GST-containing oligomers. EpsE was treated with glutathione Sepharose and applied to a Superose 6 column. Fractions of 0.5 ml were collected and analyzed by SDS-PAGE and silver staining (lower panel). These fractions were compared with the

    Techniques Used: SDS Page, Silver Staining

    Purification of EpsE. GST-EpsE was affinity purified from soluble E . coli extract with glutathione Sepharose. EpsE was cleaved from GST by the addition of thrombin (100 U/ml). Purified material was passed over benzamidine Sepharose for removal of thrombin.
    Figure Legend Snippet: Purification of EpsE. GST-EpsE was affinity purified from soluble E . coli extract with glutathione Sepharose. EpsE was cleaved from GST by the addition of thrombin (100 U/ml). Purified material was passed over benzamidine Sepharose for removal of thrombin.

    Techniques Used: Purification, Affinity Purification

    6) Product Images from "UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II"

    Article Title: UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200404045

    Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST- cdc4 and pGST- rlc1 . The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . The steps are the total cell extract after centrifuging lysed cells at 100,000 g , proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl 2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.
    Figure Legend Snippet: Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST- cdc4 and pGST- rlc1 . The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . The steps are the total cell extract after centrifuging lysed cells at 100,000 g , proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl 2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.

    Techniques Used: Purification, SDS Page, Staining, Affinity Purification, Filtration, Chromatography, Binding Assay, Solubility, Concentration Assay, Bradford Protein Assay

    Rng3p stimulates Myo2 activity. (A) Lanes 1–5 show samples from steps in the purification of Myo2 from a strain (MLP 676) containing a chromosomal rng3-GFP 3 fusion overexpressing Myo2p from the 41nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . Samples were run on an SDS-PAGE gel, immunoblotted and probed with Myo2 heavy chain antibodies (top) and GFP antibodies (bottom). (B) Bead binding assay for interaction of chromosomal Rng3p-GST with Myo2. Strains: FY 435 carrying pGST- rlc1 , MLP 694 ( rng3-GST ), MLP 693 ( rng3-GST , myo2-E1 ), and MLP 695 (wild-type). GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain (top) and Rng3p-GST (α-GST; bottom). pGST- rlc1 , positive control with GST-Rlc1p. wild type, negative control lacking a GST fusion. (C) Bead binding assay for interaction of overexpressed GST-Rng3p (pGST- rng3 - FL ) and GST-Rng3-UCS domain (pGST- rng3 - UCS ) with Myo2. Strains: FY 435 (wild-type) and TP 73 ( myo2-E1 ) carrying plasmids pGST- rng3 , pGST- rng3-UCS , pGST- cam1 , and pGST- rlc1 . GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain. pGST- cam1 , negative control with GST-Cam1p. pGST- rlc1 , positive control with GST-Rlc1p. (D) GST-Rng3p purified from S. pombe and recombinant GST-Rng3p purified from Escherichia coli . SDS-PAGE gel stained with Coomassie blue. Lower band in the S. pombe lane represents a breakdown product. (E–J) Actin filament gliding assays. Time-lapse fluorescence micrographs of filaments labeled with rhodamine-phalloidin (also, see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Bar, 5 μm. Conditions: indicated concentrations of Myo2 and GST-Rng3p were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Crude one-step purified Myo2 (0.25 mg/ml impure protein). (F) Three-step–purified 75 nM Myo2 preincubated with 250 nM native S. pombe GST-Rng3p. (G) Three-step–purified 75 nM Myo2 preincubated with 250 nM recombinant GST-Rng3p. (H) Three-step–purified 75 nM Myo2 alone. (I) S. pombe GST-Rng3p (250 nM) alone. (J) recombinant GST-Rng3p (250 nM) alone. (K) Dependence of the actin-activated ATPase activity of three-step–purified Myo2 as a function of the concentrations of native (open circles) and recombinant (closed circles) GST-Rng3p. Conditions: 30 nM Myo2 and 10 μM actin filaments in 2 mM ATP, 3 mM MgCl 2 , 0.1 mM CaCl 2 , and 75 mM KCl. Error bars show SD. (L) Dependence of the number of actin filaments captured by two-step–purified Myo2 on the concentration of recombinant GST-Rng3p. All filaments in a 130 μm 2 frame of a fluorescence micrograph were counted. (squares) 20 nM two-step–purified Myo2. No gliding was observed in the absence of GST-Rng3p. (circles) 200 nM two-step–purified Myo2. These samples supported gliding in the absence of GST-Rng3p. (horizontal line) 150 nM crude Myo2 purified after overexpression from MLP 374 ( 3nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). These samples supported robust gliding.
    Figure Legend Snippet: Rng3p stimulates Myo2 activity. (A) Lanes 1–5 show samples from steps in the purification of Myo2 from a strain (MLP 676) containing a chromosomal rng3-GFP 3 fusion overexpressing Myo2p from the 41nmt1 promoter and GST-tagged light chains from pGST- cdc4 and pGST- rlc1 . Samples were run on an SDS-PAGE gel, immunoblotted and probed with Myo2 heavy chain antibodies (top) and GFP antibodies (bottom). (B) Bead binding assay for interaction of chromosomal Rng3p-GST with Myo2. Strains: FY 435 carrying pGST- rlc1 , MLP 694 ( rng3-GST ), MLP 693 ( rng3-GST , myo2-E1 ), and MLP 695 (wild-type). GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain (top) and Rng3p-GST (α-GST; bottom). pGST- rlc1 , positive control with GST-Rlc1p. wild type, negative control lacking a GST fusion. (C) Bead binding assay for interaction of overexpressed GST-Rng3p (pGST- rng3 - FL ) and GST-Rng3-UCS domain (pGST- rng3 - UCS ) with Myo2. Strains: FY 435 (wild-type) and TP 73 ( myo2-E1 ) carrying plasmids pGST- rng3 , pGST- rng3-UCS , pGST- cam1 , and pGST- rlc1 . GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain. pGST- cam1 , negative control with GST-Cam1p. pGST- rlc1 , positive control with GST-Rlc1p. (D) GST-Rng3p purified from S. pombe and recombinant GST-Rng3p purified from Escherichia coli . SDS-PAGE gel stained with Coomassie blue. Lower band in the S. pombe lane represents a breakdown product. (E–J) Actin filament gliding assays. Time-lapse fluorescence micrographs of filaments labeled with rhodamine-phalloidin (also, see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Bar, 5 μm. Conditions: indicated concentrations of Myo2 and GST-Rng3p were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Crude one-step purified Myo2 (0.25 mg/ml impure protein). (F) Three-step–purified 75 nM Myo2 preincubated with 250 nM native S. pombe GST-Rng3p. (G) Three-step–purified 75 nM Myo2 preincubated with 250 nM recombinant GST-Rng3p. (H) Three-step–purified 75 nM Myo2 alone. (I) S. pombe GST-Rng3p (250 nM) alone. (J) recombinant GST-Rng3p (250 nM) alone. (K) Dependence of the actin-activated ATPase activity of three-step–purified Myo2 as a function of the concentrations of native (open circles) and recombinant (closed circles) GST-Rng3p. Conditions: 30 nM Myo2 and 10 μM actin filaments in 2 mM ATP, 3 mM MgCl 2 , 0.1 mM CaCl 2 , and 75 mM KCl. Error bars show SD. (L) Dependence of the number of actin filaments captured by two-step–purified Myo2 on the concentration of recombinant GST-Rng3p. All filaments in a 130 μm 2 frame of a fluorescence micrograph were counted. (squares) 20 nM two-step–purified Myo2. No gliding was observed in the absence of GST-Rng3p. (circles) 200 nM two-step–purified Myo2. These samples supported gliding in the absence of GST-Rng3p. (horizontal line) 150 nM crude Myo2 purified after overexpression from MLP 374 ( 3nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). These samples supported robust gliding.

    Techniques Used: Activity Assay, Purification, SDS Page, Binding Assay, Affinity Purification, Positive Control, Negative Control, Recombinant, Staining, Fluorescence, Labeling, Flow Cytometry, Concentration Assay, Over Expression

    Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4 , rlc1 , cam1 , or cam2 . The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plu s YFP-cdc4 , myo2-GST plus rlc1-GFP , or myo2-GST plus cam2-GFP . Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.
    Figure Legend Snippet: Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4 , rlc1 , cam1 , or cam2 . The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plu s YFP-cdc4 , myo2-GST plus rlc1-GFP , or myo2-GST plus cam2-GFP . Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.

    Techniques Used: SDS Page, Affinity Purification, Expressing

    Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH 2 -terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1 Δ strain (MLP 7) carrying empty vector (negative control), pGFP- rlc1 (positive control), pGFP- rlc1-N1 Δ, or pGFP- rlc1-N2 Δ. Transformants were streaked on an EMM Ura - plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP- rlc1 (positive control), or pGFP- rlc1-N1 Δ. Transformants were grown in liquid EMM Ura − media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP- rlc1 (left panels) or pGFP- rlc1-N1 Δ (right panels). Cells were grown in liquid EMM Ura − media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1 Δ 41nmt1 promoter- myo2 plus pGST- cdc4 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6- s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 ( 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter- myo2 , plus pGST- cdc4-8 and pGST- rlc1 ; MLP 641 cdc4-31 41nmt1 promoter- myo2 plus pGST- cdc4-31 and pGST- rlc1 ; MLP 647 cdc4-C2 41nmt1 promoter- myo2 plus pGST- cdc4-C2 and pGST- rlc1 ; MLP 648 cdc4-A2 41nmt1 promoter- myo2 plus pGST- cdc4-A2 and pGST- rlc1 .
    Figure Legend Snippet: Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH 2 -terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1 Δ strain (MLP 7) carrying empty vector (negative control), pGFP- rlc1 (positive control), pGFP- rlc1-N1 Δ, or pGFP- rlc1-N2 Δ. Transformants were streaked on an EMM Ura - plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP- rlc1 (positive control), or pGFP- rlc1-N1 Δ. Transformants were grown in liquid EMM Ura − media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP- rlc1 (left panels) or pGFP- rlc1-N1 Δ (right panels). Cells were grown in liquid EMM Ura − media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl 2 , 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1 Δ 41nmt1 promoter- myo2 plus pGST- cdc4 ). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6- s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 ( 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter- myo2 plus pGST- cdc4 and pGST- rlc1 ). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter- myo2 , plus pGST- cdc4-8 and pGST- rlc1 ; MLP 641 cdc4-31 41nmt1 promoter- myo2 plus pGST- cdc4-31 and pGST- rlc1 ; MLP 647 cdc4-C2 41nmt1 promoter- myo2 plus pGST- cdc4-C2 and pGST- rlc1 ; MLP 648 cdc4-A2 41nmt1 promoter- myo2 plus pGST- cdc4-A2 and pGST- rlc1 .

    Techniques Used: Activity Assay, Sequencing, Generated, Software, Plasmid Preparation, Negative Control, Positive Control, Quantitation Assay, Staining, Fluorescence, Microscopy, Isolation, Labeling, Flow Cytometry, Purification, Mutagenesis, In Vitro

    7) Product Images from "14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival"

    Article Title: 14-3-3:Shc Scaffolds Integrate Phosphoserine and Phosphotyrosine Signaling to Regulate Phosphatidylinositol 3-Kinase Activation and Cell Survival

    Journal:

    doi: 10.1074/jbc.M807637200

    14-3-3 proteins undergo tyrosine phosphorylation in response to GM-CSF stimulation. A , primary human mononuclear cells from peripheral blood ( left panel ) or CTL-EN cells ( right panel ) were stimulated with GM-CSF. Endogenous 14-3-3 was precipitated with a Ser(P)585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG; to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-Shc and anti-14-3-3 pAbs. B , factor-dependent CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight in medium containing 0.5% FCS and then stimulated with GM-CSF. The cells were lysed, and pulldown experiments were performed using either GST-SH2Shc (to precipitate tyrosine-phosphorylated endogenous 14-3-3) or the Ser(P)585 peptide (to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-14-3-3 pAb. C , HEK 293T cells were transfected with a construct for the expression of wt 14-3-3ζ-Myc. After 48 h, the cells were lysed and 14-3-3ζ-Myc immunoprecipitated using the 9E10 mAb. Immunoprecipitates were subjected to immunoblot analysis with either the 4G10 mAb (Millipore) ( top panel ), an anti-phosphotyrosine pAb (Biomol) ( middle panel ) or the 9E10 mAb ( bottom panel ). D , the crystal structure of human 14-3-3ζ indicates that it is composed of 2 monomers that dimerize to form a large central groove that binds to phosphoserine peptides (PS-peptide) . The location of tyrosines 19, 118, 178, 179, and 211 of 14-3-3ζ are indicated. E and F , recombinant purified 14-3-3ζ and 14-3-3ζ-T179F were phosphorylated in vitro using c-Src and then subjected to immunoblot analysis using the 4G10 anti-phosphotyrosine mAb ( anti-PY ) ( E, top panel ), anti-Tyr(P)179 -14-3-3ζ pAb ( anti-PY179 ) ( F, top panel ) or anti-14-3-3ζ pAb to indicate loading ( E and F, bottom panels ). G , HEK-293T cells were transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were stimulated for 15 min with sodium pervanadate (VO4 ) (+) or left unstimulated (-). The cells were then lysed and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to immunoblot analysis using the anti-Tyr(P)179 -14-3-3ζ pAb ( G, top panel ) and the 9E10 mAb ( G, bottom panel ). H , CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF or sodium pervanadate (VO4 ), following which the cells were lysed, and endogenous 14-3-3 was precipitated ( PD ) using either a Ser(P)585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or an Ser585 (non-phospho-Ser585 ) control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose resin. Pulldowns were subjected to immunoblot analysis using the anti-Tyr(P)179 -14-3-3ζ pAb or anti-14-3-3 pAb. I , primary human mononuclear cells were stimulated with GM-CSF and total endogenous 14-3-3 was precipitated as in A . Precipitates were blotted with anti-Tyr(P)179 -14-3-3ζ pAb or anti-14-3-3 pAb. J , the UT7 factor-dependent cell line was factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF, following which the cells were lysed, and the βc subunit of the GM-CSF receptor was immunoprecipitated using the 8E4 and 1C1 mAbs. Immunoprecipitates were then subjected to immunoblot analysis with either anti-Tyr(P)179 pAb ( top panel ) or 1C1 anti-βc mAb ( bottom panel ). The results are typical of at least two experiments. WB , Western blotting.
    Figure Legend Snippet: 14-3-3 proteins undergo tyrosine phosphorylation in response to GM-CSF stimulation. A , primary human mononuclear cells from peripheral blood ( left panel ) or CTL-EN cells ( right panel ) were stimulated with GM-CSF. Endogenous 14-3-3 was precipitated with a Ser(P)585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG; to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-Shc and anti-14-3-3 pAbs. B , factor-dependent CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight in medium containing 0.5% FCS and then stimulated with GM-CSF. The cells were lysed, and pulldown experiments were performed using either GST-SH2Shc (to precipitate tyrosine-phosphorylated endogenous 14-3-3) or the Ser(P)585 peptide (to precipitate total endogenous 14-3-3), and precipitates were immunoblotted with anti-14-3-3 pAb. C , HEK 293T cells were transfected with a construct for the expression of wt 14-3-3ζ-Myc. After 48 h, the cells were lysed and 14-3-3ζ-Myc immunoprecipitated using the 9E10 mAb. Immunoprecipitates were subjected to immunoblot analysis with either the 4G10 mAb (Millipore) ( top panel ), an anti-phosphotyrosine pAb (Biomol) ( middle panel ) or the 9E10 mAb ( bottom panel ). D , the crystal structure of human 14-3-3ζ indicates that it is composed of 2 monomers that dimerize to form a large central groove that binds to phosphoserine peptides (PS-peptide) . The location of tyrosines 19, 118, 178, 179, and 211 of 14-3-3ζ are indicated. E and F , recombinant purified 14-3-3ζ and 14-3-3ζ-T179F were phosphorylated in vitro using c-Src and then subjected to immunoblot analysis using the 4G10 anti-phosphotyrosine mAb ( anti-PY ) ( E, top panel ), anti-Tyr(P)179 -14-3-3ζ pAb ( anti-PY179 ) ( F, top panel ) or anti-14-3-3ζ pAb to indicate loading ( E and F, bottom panels ). G , HEK-293T cells were transfected with constructs for the expression of wt 14-3-3ζ-Myc or the T179F mutant. After 48 h, the cells were stimulated for 15 min with sodium pervanadate (VO4 ) (+) or left unstimulated (-). The cells were then lysed and subjected to immunoprecipitation using the 9E10 mAb. Immunoprecipitated proteins were subjected to immunoblot analysis using the anti-Tyr(P)179 -14-3-3ζ pAb ( G, top panel ) and the 9E10 mAb ( G, bottom panel ). H , CTL-EN cells expressing the human GM-CSF receptor were factor-deprived overnight. The cells were then stimulated with 50 ng/ml GM-CSF or sodium pervanadate (VO4 ), following which the cells were lysed, and endogenous 14-3-3 was precipitated ( PD ) using either a Ser(P)585 peptide (Biotin-NHS-KGGFDFNGPYLGPPHSR(pS)LPDGG) or an Ser585 (non-phospho-Ser585 ) control peptide (Biotin-NHS-KGGFDFNGPYLGPPHSRSLPDGG) adsorbed to streptavidin-Sepharose resin. Pulldowns were subjected to immunoblot analysis using the anti-Tyr(P)179 -14-3-3ζ pAb or anti-14-3-3 pAb. I , primary human mononuclear cells were stimulated with GM-CSF and total endogenous 14-3-3 was precipitated as in A . Precipitates were blotted with anti-Tyr(P)179 -14-3-3ζ pAb or anti-14-3-3 pAb. J , the UT7 factor-dependent cell line was factor-deprived overnight and then stimulated with 50 ng/ml GM-CSF, following which the cells were lysed, and the βc subunit of the GM-CSF receptor was immunoprecipitated using the 8E4 and 1C1 mAbs. Immunoprecipitates were then subjected to immunoblot analysis with either anti-Tyr(P)179 pAb ( top panel ) or 1C1 anti-βc mAb ( bottom panel ). The results are typical of at least two experiments. WB , Western blotting.

    Techniques Used: CTL Assay, Expressing, Transfection, Construct, Immunoprecipitation, Recombinant, Purification, In Vitro, Mutagenesis, Western Blot

    8) Product Images from "The Drosophila Importin-?3 Is Required for Nuclear Import of Notch In Vivo and It Displays Synergistic Effects with Notch Receptor on Cell Proliferation"

    Article Title: The Drosophila Importin-?3 Is Required for Nuclear Import of Notch In Vivo and It Displays Synergistic Effects with Notch Receptor on Cell Proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068247

    Drosophila Notch binds Importin-α3. (A) Schematic representation of the domain organization of Importin-α3. Different domains and boundary residues are marked on top. IBB, Importin β binding domain; ARM, Armadillo repeats [see refs 19, 20]. A region of Importin-α3 (amino acids 240–502) that was sufficient for binding to Notch, based on yeast two-hybrid analysis, is shown below the full-length protein. (B) GST-pulldown assay was performed with lysate of salivary glands in which Notch-ICD was overexpressed using salivary gland specific GAL4 driver ( sgs-GAL4 ) and purified recombinant GST-Importin-α3 full-length (amino acids 1–514), amino-terminal (amino acids 1–224), carboxy-terminal (amino acids 225–514) and other controls as indicated. GST pulled down proteins were analyzed by western blotting with anti-Notch (C17.9C6) antibodies. GST-Importin-α3 full-length and GST-Importin-α3 carboxy-terminus pulled down Notch-ICD. (C) Co-immunoprecipitation of HA-Importin-α3 and Notch-ICD. HA-Importin-α3 and Notch-ICD were co-expressed in larval salivary glands and immunoprecipitated with anti-HA agarose. Immunoprecipitated proteins were analyzed by western blotting with anti-Notch (C17.9C6) antibodies (upper panel) and with anti-HA antibodies (lower panel). Middle panel shows the level of Notch protein in the lysates. (D1–F4) Co-localization of HA-Importin-α3 and Notch-ICD in salivary glands (D1–E4) and eye discs (F1–F4). UAS-HA-imp-α3 and UAS-Notch-ICD were expressed under the control of the ey-GAL4 driver. Images in D4, E4, and F4 are merges of those in D1–D3, E1–E3, and F1–F3, respectively. Images in E1–E4 are high magnification images of a single cell from salivary glands shown in D1–D4. Co-expression of HA-Importin-α3 and Notch-ICD shows their co-localization in cell nuclei (arrowheads). Scale bars, 100 µm (D1–D4), 10 µm (E1–F4).
    Figure Legend Snippet: Drosophila Notch binds Importin-α3. (A) Schematic representation of the domain organization of Importin-α3. Different domains and boundary residues are marked on top. IBB, Importin β binding domain; ARM, Armadillo repeats [see refs 19, 20]. A region of Importin-α3 (amino acids 240–502) that was sufficient for binding to Notch, based on yeast two-hybrid analysis, is shown below the full-length protein. (B) GST-pulldown assay was performed with lysate of salivary glands in which Notch-ICD was overexpressed using salivary gland specific GAL4 driver ( sgs-GAL4 ) and purified recombinant GST-Importin-α3 full-length (amino acids 1–514), amino-terminal (amino acids 1–224), carboxy-terminal (amino acids 225–514) and other controls as indicated. GST pulled down proteins were analyzed by western blotting with anti-Notch (C17.9C6) antibodies. GST-Importin-α3 full-length and GST-Importin-α3 carboxy-terminus pulled down Notch-ICD. (C) Co-immunoprecipitation of HA-Importin-α3 and Notch-ICD. HA-Importin-α3 and Notch-ICD were co-expressed in larval salivary glands and immunoprecipitated with anti-HA agarose. Immunoprecipitated proteins were analyzed by western blotting with anti-Notch (C17.9C6) antibodies (upper panel) and with anti-HA antibodies (lower panel). Middle panel shows the level of Notch protein in the lysates. (D1–F4) Co-localization of HA-Importin-α3 and Notch-ICD in salivary glands (D1–E4) and eye discs (F1–F4). UAS-HA-imp-α3 and UAS-Notch-ICD were expressed under the control of the ey-GAL4 driver. Images in D4, E4, and F4 are merges of those in D1–D3, E1–E3, and F1–F3, respectively. Images in E1–E4 are high magnification images of a single cell from salivary glands shown in D1–D4. Co-expression of HA-Importin-α3 and Notch-ICD shows their co-localization in cell nuclei (arrowheads). Scale bars, 100 µm (D1–D4), 10 µm (E1–F4).

    Techniques Used: Binding Assay, GST Pulldown Assay, Purification, Recombinant, Western Blot, Immunoprecipitation, Hemagglutination Assay, Expressing

    9) Product Images from "SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation"

    Article Title: SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx031

    Reconstitution and activity of the SBP2/methylosome complex and functional analysis ( A ) Coexpression and purification of SBP2 associated with the methylosome complex. Recombinant baculoviruses allowing the expression of GST-SBP2, PRMT5, MEP50 and HA-pICln were used to infect Sf9 insect cells in the combinations indicated. Expression of PRMT5/MEP50 and HA-pICln alone served as negative controls. The complexes associated to GST-SBP2 were purified on Glutathione Sepharose, and the associated proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (4% of the total cell extract). ( B ) SBP2 is not a substrate of PRMT5/MEP50. For methylation assays PRMT5/MEP50 was incubated with SBP2 or histones (H3-H4) 2 in the presence of 14 C SAM for 30 min at 37°C. The (H3-H4) 2 tetramer is a methylation substrate of the methylosome and therefore serves as a positive control. Proteins are analyzed on SDS-PAGE and revealed by coomassie staining (lanes 1–3). The radioactive methylation signal is revealed by phosphorImager after overnight exposure (lanes 4–6). The cartoon represents His-Tag SBP2 and the position of the RG sequences (*) that are potential sites of methylation. L: ladder. ( C ) Sm proteins do not bind selenoprotein mRNAs. Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-Sm antibodies. qRT-PCR was used to determine the RNA enrichment in the IP experiment compared to the input extract by the ΔΔCt method. SelR, GPx4, SelM, SelT, TrxR1 and Sel15 are selenoprotein mRNAs. U1 and U2 snRNAs were used as positive controls; ß-actin and LDHA mRNAs are housekeeping mRNAs used as negative controls. Error bars represent standard deviation of an average of three independent experiments.
    Figure Legend Snippet: Reconstitution and activity of the SBP2/methylosome complex and functional analysis ( A ) Coexpression and purification of SBP2 associated with the methylosome complex. Recombinant baculoviruses allowing the expression of GST-SBP2, PRMT5, MEP50 and HA-pICln were used to infect Sf9 insect cells in the combinations indicated. Expression of PRMT5/MEP50 and HA-pICln alone served as negative controls. The complexes associated to GST-SBP2 were purified on Glutathione Sepharose, and the associated proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (4% of the total cell extract). ( B ) SBP2 is not a substrate of PRMT5/MEP50. For methylation assays PRMT5/MEP50 was incubated with SBP2 or histones (H3-H4) 2 in the presence of 14 C SAM for 30 min at 37°C. The (H3-H4) 2 tetramer is a methylation substrate of the methylosome and therefore serves as a positive control. Proteins are analyzed on SDS-PAGE and revealed by coomassie staining (lanes 1–3). The radioactive methylation signal is revealed by phosphorImager after overnight exposure (lanes 4–6). The cartoon represents His-Tag SBP2 and the position of the RG sequences (*) that are potential sites of methylation. L: ladder. ( C ) Sm proteins do not bind selenoprotein mRNAs. Total RNA extracted from HEK293FT cells was immunoprecipitated with anti-Sm antibodies. qRT-PCR was used to determine the RNA enrichment in the IP experiment compared to the input extract by the ΔΔCt method. SelR, GPx4, SelM, SelT, TrxR1 and Sel15 are selenoprotein mRNAs. U1 and U2 snRNAs were used as positive controls; ß-actin and LDHA mRNAs are housekeeping mRNAs used as negative controls. Error bars represent standard deviation of an average of three independent experiments.

    Techniques Used: Activity Assay, Functional Assay, Purification, Recombinant, Expressing, Hemagglutination Assay, SDS Page, Western Blot, Methylation, Incubation, Positive Control, Staining, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    The methylosome complex interacts with SBP2 in vivo . ( A ) Immunopurification of endogenous SBP2 from HeLa cytoplasmic extracts using antipeptide antibodies (α-pepSBP2) directed against SBP2 residues 380–852. PI: beads with preimmune serum. The immunopurified proteins were identified by mass spectrometry (MS) and specific proteins are indicated on the right. Numbers represent common sepharose-matrix binding contaminants found in MS analysis ( 56 ), most of them are cytoskeletal proteins (1,1΄: Dynein chains, 2: Keratin, 3: Tubulin ß; 4: Tubulin α; 5: VASP actin associated protein). Molecular weight (kDa) is indicated. ( B–D ) Co-immunoprecipitations using anti-GFP beads and HEK293FT cells transfected by (B and C) GFP-SBP2 and (D) GFP-PRMT5, GFP-MEP50, GFP-pICln or GFP alone. Immunoprecipitations were performed in high salt (300 mM NaCl) and low salt (150 mM NaCl) conditions. The immunoprecipitated endogenous proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (10% of total). * smear of unknown origin always present at high salt but absent at low salt. ( E ) Direct interactions between SBP2 and components of the methylosome complex. Y2H interaction tests performed in Saccharomyces cerevisiae AH109 between SBP2 and the methylosome components PRMT5, MEP50 and pICln; Nufip was used as a positive interaction control with SBP2. Experiments were performed as described Figure 1B . Data are shown in Supplementary Figure S1A .
    Figure Legend Snippet: The methylosome complex interacts with SBP2 in vivo . ( A ) Immunopurification of endogenous SBP2 from HeLa cytoplasmic extracts using antipeptide antibodies (α-pepSBP2) directed against SBP2 residues 380–852. PI: beads with preimmune serum. The immunopurified proteins were identified by mass spectrometry (MS) and specific proteins are indicated on the right. Numbers represent common sepharose-matrix binding contaminants found in MS analysis ( 56 ), most of them are cytoskeletal proteins (1,1΄: Dynein chains, 2: Keratin, 3: Tubulin ß; 4: Tubulin α; 5: VASP actin associated protein). Molecular weight (kDa) is indicated. ( B–D ) Co-immunoprecipitations using anti-GFP beads and HEK293FT cells transfected by (B and C) GFP-SBP2 and (D) GFP-PRMT5, GFP-MEP50, GFP-pICln or GFP alone. Immunoprecipitations were performed in high salt (300 mM NaCl) and low salt (150 mM NaCl) conditions. The immunoprecipitated endogenous proteins were analyzed by SDS-PAGE and western blotting using the indicated antibodies. In: input (10% of total). * smear of unknown origin always present at high salt but absent at low salt. ( E ) Direct interactions between SBP2 and components of the methylosome complex. Y2H interaction tests performed in Saccharomyces cerevisiae AH109 between SBP2 and the methylosome components PRMT5, MEP50 and pICln; Nufip was used as a positive interaction control with SBP2. Experiments were performed as described Figure 1B . Data are shown in Supplementary Figure S1A .

    Techniques Used: In Vivo, Immu-Puri, Mass Spectrometry, Binding Assay, Molecular Weight, Transfection, Immunoprecipitation, SDS Page, Western Blot

    10) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M112.368654

    STEP dephosphorylates Pyk2 at Tyr 402 . A , Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate ( Perv ) (1 m m ). Mg-ATP, STEP (10 μ m ), and pervanadate (1 m m ) were added
    Figure Legend Snippet: STEP dephosphorylates Pyk2 at Tyr 402 . A , Pyk2 was immunoprecipitated from mouse brain lysates using anti-Pyk2 antibody and protein A/G-agarose in the presence of pervanadate ( Perv ) (1 m m ). Mg-ATP, STEP (10 μ m ), and pervanadate (1 m m ) were added

    Techniques Used: Immunoprecipitation

    Phosphorylation of Pyk2 at Tyr 402 is increased in presence of protein-tyrosine phosphatase inhibitor. A , rat brain extracts were incubated with 1 μg of αN-Pyk2 antibody in the absence of protein A-agarose. Mg-ATP was then added in the
    Figure Legend Snippet: Phosphorylation of Pyk2 at Tyr 402 is increased in presence of protein-tyrosine phosphatase inhibitor. A , rat brain extracts were incubated with 1 μg of αN-Pyk2 antibody in the absence of protein A-agarose. Mg-ATP was then added in the

    Techniques Used: Incubation

    11) Product Images from "O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release"

    Article Title: O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2012.8

    TAB1 is O -GlcNAcylated on Ser395. ( A ) Recombinant TAB1 was O -GlcNAcylated in vitro with recombinant hOGT. The samples were denatured in LDS (lithium dodecyl sulphate), subjected to SDS–PAGE and immunoblotted with a generic O -GlcNAc antibody CTD110.6. Total protein was detected with a total TAB1 antibody (TAB1) as a loading control. ( B ) In vitro O -GlcNAcylated TAB1 was subjected to enzymatic labelling using galactosyltransferase (mGalT1) labelling and UDP-GalNAz, before reacting with biotin alkyne for detection with HRP. The samples were denatured in LDS, subjected to SDS–PAGE and probed with horseradish peroxidase-conjugated streptavidin (Extravidin-HRP). ( C ) In vivo O -GlcNAcylation of TAB1 was detected by immunoprecipitating the endogenous TAB1 from IL-1R cells (treated with or without GlcNAcstatin—1 μM) using an antibody directed against TAB1. A non-specific IgG was included as a control. Immunoprecipitates were denatured in LDS, subjected to SDS–PAGE and immunoblotted with generic O -GlcNAc antibody CTD110.6 and then with the TAB1 antibody for loading controls. Treating the cells with the potent and selective OGA inhibitor GlcNAcstatin further increased TAB1 O -GlcNAcylation at a concentration of 1 μM. ( D ) TAB1 was immunoprecipitated, from cells treated with or without GlcNAcstatin (1 μM), using the TAB1 antibody. One fraction of the reaction was treated with Cp OGA for 30 min at room temperature, denatured in LDS, followed by immunoblotting with the generic O -GlcNAc antibody CTD110.6. A parallel set of immunoprecipitated samples were denatured in LDS and subjected to western blotting with antibody pre-incubated with N -acetylglucosamine, which blocks the O -GlcNAc signal on TAB1. Lower panel shows the corresponding Ponceau-stained membrane before western blotting. ( E ) WT TAB1, the S391A/S395A/S396A TAB1 triple mutant and the S391A, S395A and S396A TAB1 single mutants were transfected into IL-1R cells. After 24 h, the cells were treated with 1 μM of GlcNAcstatin for 16 h and the GSt–TAB1 was pulled out using glutathione-sepharose beads. The samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with the generic O -GlcNAc antibody CTD110.6 and then with a total TAB1 antibody (TAB1) as a loading control. ( F , G ) LC-MS/MS CID ( F ) and ETD ( G ) site mapping of the TAB1 O -GlcNAc modification site. In vitro O-GlcNAcylated TAB1 was digested with trypsin and subjected to LC–MS. The tryptic peptide VYPVSVPYSSAQSTSK (Mw calc =1901.9 Da) containing a HexNAc (+203.1 Da) was detected after 25.5 min as [M+HexNAc+2H] 2+ m / z 951.904. The observed fragment ions are indicated, in case of the ETD experiment allowing definition of S395 as the site of O -GlcNAc modification. Figure source data can be found in Supplementary data .
    Figure Legend Snippet: TAB1 is O -GlcNAcylated on Ser395. ( A ) Recombinant TAB1 was O -GlcNAcylated in vitro with recombinant hOGT. The samples were denatured in LDS (lithium dodecyl sulphate), subjected to SDS–PAGE and immunoblotted with a generic O -GlcNAc antibody CTD110.6. Total protein was detected with a total TAB1 antibody (TAB1) as a loading control. ( B ) In vitro O -GlcNAcylated TAB1 was subjected to enzymatic labelling using galactosyltransferase (mGalT1) labelling and UDP-GalNAz, before reacting with biotin alkyne for detection with HRP. The samples were denatured in LDS, subjected to SDS–PAGE and probed with horseradish peroxidase-conjugated streptavidin (Extravidin-HRP). ( C ) In vivo O -GlcNAcylation of TAB1 was detected by immunoprecipitating the endogenous TAB1 from IL-1R cells (treated with or without GlcNAcstatin—1 μM) using an antibody directed against TAB1. A non-specific IgG was included as a control. Immunoprecipitates were denatured in LDS, subjected to SDS–PAGE and immunoblotted with generic O -GlcNAc antibody CTD110.6 and then with the TAB1 antibody for loading controls. Treating the cells with the potent and selective OGA inhibitor GlcNAcstatin further increased TAB1 O -GlcNAcylation at a concentration of 1 μM. ( D ) TAB1 was immunoprecipitated, from cells treated with or without GlcNAcstatin (1 μM), using the TAB1 antibody. One fraction of the reaction was treated with Cp OGA for 30 min at room temperature, denatured in LDS, followed by immunoblotting with the generic O -GlcNAc antibody CTD110.6. A parallel set of immunoprecipitated samples were denatured in LDS and subjected to western blotting with antibody pre-incubated with N -acetylglucosamine, which blocks the O -GlcNAc signal on TAB1. Lower panel shows the corresponding Ponceau-stained membrane before western blotting. ( E ) WT TAB1, the S391A/S395A/S396A TAB1 triple mutant and the S391A, S395A and S396A TAB1 single mutants were transfected into IL-1R cells. After 24 h, the cells were treated with 1 μM of GlcNAcstatin for 16 h and the GSt–TAB1 was pulled out using glutathione-sepharose beads. The samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with the generic O -GlcNAc antibody CTD110.6 and then with a total TAB1 antibody (TAB1) as a loading control. ( F , G ) LC-MS/MS CID ( F ) and ETD ( G ) site mapping of the TAB1 O -GlcNAc modification site. In vitro O-GlcNAcylated TAB1 was digested with trypsin and subjected to LC–MS. The tryptic peptide VYPVSVPYSSAQSTSK (Mw calc =1901.9 Da) containing a HexNAc (+203.1 Da) was detected after 25.5 min as [M+HexNAc+2H] 2+ m / z 951.904. The observed fragment ions are indicated, in case of the ETD experiment allowing definition of S395 as the site of O -GlcNAc modification. Figure source data can be found in Supplementary data .

    Techniques Used: Recombinant, In Vitro, SDS Page, In Vivo, Concentration Assay, Immunoprecipitation, Western Blot, Incubation, Staining, Mutagenesis, Transfection, Liquid Chromatography, Mass Spectrometry, Modification

    O -GlcNAcylation of TAB1 affects activation of TAK1 and phosphorylation of its downstream targets IκBα and JNK1/2. ( A ) IL-1α or NaCl-induced activation of TAK1 in WT and S395A TAB1 transfected Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 and 15 min with 10 ng/ml IL-1α or 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads, and TAK1 activity assays were performed (as described in the Materials and methods section) in addition to immunoblotting as described below in ( B ). The data are expressed as the relative increase in TAK1 activity of the IL-1/NaCl-stimulated samples compared with the basal activity of the unstimulated control samples. Error bars denote standard deviation, determined from three independent experiments. ( B ) In parallel to the experiments in ( A ), TAK1 complexes were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( C ) In all, 30 μg of the cell lysates from the samples obtained as in ( A ) was immunoblotted for phosphorylated Iκβα p-Iκβα and total Iκβα. ( D ) Densitometry for IκBα phosphorylation after normalization against total TAB1 levels. The data shown are the average of three independent experiments with error bars denoting standard deviation. ( E ) WT and S395A TAB1 were transfected in Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 or 15 min with 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads and taken for kinase assays ( A ) in addition to immunoblotting. For immunoblotting, the samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( F ) In all, 30 μg of the cell lysates from the samples obtained as in ( E ) was immunoblotted for phosphorylated JNK1/2 (p-JNK1/2) and total JNK1/2. ( G ) Densitometry for JNK1/2 phosphorylation after normalization for total JNK1/2. The data shown are the average of minimum of three independent experiments with error bars denoting standard deviation. Figure source data can be found in Supplementary data .
    Figure Legend Snippet: O -GlcNAcylation of TAB1 affects activation of TAK1 and phosphorylation of its downstream targets IκBα and JNK1/2. ( A ) IL-1α or NaCl-induced activation of TAK1 in WT and S395A TAB1 transfected Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 and 15 min with 10 ng/ml IL-1α or 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads, and TAK1 activity assays were performed (as described in the Materials and methods section) in addition to immunoblotting as described below in ( B ). The data are expressed as the relative increase in TAK1 activity of the IL-1/NaCl-stimulated samples compared with the basal activity of the unstimulated control samples. Error bars denote standard deviation, determined from three independent experiments. ( B ) In parallel to the experiments in ( A ), TAK1 complexes were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( C ) In all, 30 μg of the cell lysates from the samples obtained as in ( A ) was immunoblotted for phosphorylated Iκβα p-Iκβα and total Iκβα. ( D ) Densitometry for IκBα phosphorylation after normalization against total TAB1 levels. The data shown are the average of three independent experiments with error bars denoting standard deviation. ( E ) WT and S395A TAB1 were transfected in Tab1 −/− MEFs. At 36 h post-transfection, MEFs were serum starved for 6 h, and then stimulated for 5 or 15 min with 0.5 M NaCl. The TAK1 complexes were pulled down from the cell extracts (1 mg of protein extract) using glutathione-sepharose beads and taken for kinase assays ( A ) in addition to immunoblotting. For immunoblotting, the samples were denatured in LDS, subjected to SDS–PAGE and immunoblotted with a phospho-specific antibody that recognizes TAK1 autophosphorylation at Thr187 (pT187) and with a further antibody that recognizes all forms of TAK1. O -GlcNAcylation of TAB1 was detected with the site-specific O -GlcNAc antibody (gS395) versus a total TAB1 antibody control (TAB1). ( F ) In all, 30 μg of the cell lysates from the samples obtained as in ( E ) was immunoblotted for phosphorylated JNK1/2 (p-JNK1/2) and total JNK1/2. ( G ) Densitometry for JNK1/2 phosphorylation after normalization for total JNK1/2. The data shown are the average of minimum of three independent experiments with error bars denoting standard deviation. Figure source data can be found in Supplementary data .

    Techniques Used: Activation Assay, Transfection, Activity Assay, Standard Deviation, SDS Page

    12) Product Images from "IQGAP1 Binds to Estrogen Receptor-α and Modulates Its Function"

    Article Title: IQGAP1 Binds to Estrogen Receptor-α and Modulates Its Function

    Journal:

    doi: 10.1074/jbc.M114.553511

    IQGAP1 and ERα bind directly in vitro . A , 1 μg of GST-IQGAP1 ( IQGAP1 ) or 1 μg of GST alone bound to glutathione-Sepharose was incubated with 1 μg of pure ERα. Complexes were isolated and washed as described under
    Figure Legend Snippet: IQGAP1 and ERα bind directly in vitro . A , 1 μg of GST-IQGAP1 ( IQGAP1 ) or 1 μg of GST alone bound to glutathione-Sepharose was incubated with 1 μg of pure ERα. Complexes were isolated and washed as described under

    Techniques Used: In Vitro, Incubation, Isolation

    ERα and IQGAP1 interact in cells. A , 4 μg of GST-IQGAP1 ( IQGAP1 ) or 4 μg of GST alone bound to glutathione-Sepharose was incubated with lysates from MCF-7 ( left panel ) or T47D ( right panel ) breast epithelial cells. Complexes were
    Figure Legend Snippet: ERα and IQGAP1 interact in cells. A , 4 μg of GST-IQGAP1 ( IQGAP1 ) or 4 μg of GST alone bound to glutathione-Sepharose was incubated with lysates from MCF-7 ( left panel ) or T47D ( right panel ) breast epithelial cells. Complexes were

    Techniques Used: Incubation

    13) Product Images from "SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase"

    Article Title: SH3-binding Protein 5 Mediates the Neuroprotective Effect of the Secreted Bioactive Peptide Humanin by Inhibiting c-Jun NH2-terminal Kinase

    Journal:

    doi: 10.1074/jbc.M113.469692

    SH3BP5 is ubiquitously expressed and localized in mitochondria. A , immunohistochemical analysis of SH3BP5 expression in various mouse neuronal tissues using the SH3BP5 antibody ( green ). The SH3BP5 antibody was preincubated (preabsorbed) with GST (a negative control) or GST-SH3BP5 (immunizing protein), bound to glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ). B , F11 cells were immunostained with the SH3BP5 antibody ( green ) and the MitoTracker® ( red ). Nuclei were stained with DAPI ( blue ). The SH3BP5 antibody was preincubated (preabsorbed) with GST (a negative control) or GST-SH3BP5 (immunizing protein), bound to glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ).
    Figure Legend Snippet: SH3BP5 is ubiquitously expressed and localized in mitochondria. A , immunohistochemical analysis of SH3BP5 expression in various mouse neuronal tissues using the SH3BP5 antibody ( green ). The SH3BP5 antibody was preincubated (preabsorbed) with GST (a negative control) or GST-SH3BP5 (immunizing protein), bound to glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ). B , F11 cells were immunostained with the SH3BP5 antibody ( green ) and the MitoTracker® ( red ). Nuclei were stained with DAPI ( blue ). The SH3BP5 antibody was preincubated (preabsorbed) with GST (a negative control) or GST-SH3BP5 (immunizing protein), bound to glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ).

    Techniques Used: Immunohistochemistry, Expressing, Negative Control, Staining

    Both mRNA and protein expressions of SH3BP5 are induced by Humanin. A , real-time PCR analysis of SH3BP5 and GAPDH mRNAs, prepared from mouse PHNs and human SH-SY5Y cells, incubated with 10 μ m HN or distilled water ( DW ) for 24 h. This experiment was performed with n = 3; ***, p < 0.001; *, p < 0.05. B , F11 cells, transfected with the pCMV-human SH3BP5 vector, were immunostained with the SH3BP5 antibody ( red ), not preabsorbed ( left ), preabsorbed with GST ( middle ), or preabsorbed with GST-SH3BP5 ( right ), which had been immobilized onto glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ). C , immunocytochemical analysis of SH-SY5Y cells with the SH3BP5 antibody ( red ), incubated with 100 n m Humanin G ( HNG ) or distilled water ( DW ) in the presence or the absence of 5 μ m AG490 for 24 h. Nuclei were stained with DAPI ( blue ).
    Figure Legend Snippet: Both mRNA and protein expressions of SH3BP5 are induced by Humanin. A , real-time PCR analysis of SH3BP5 and GAPDH mRNAs, prepared from mouse PHNs and human SH-SY5Y cells, incubated with 10 μ m HN or distilled water ( DW ) for 24 h. This experiment was performed with n = 3; ***, p < 0.001; *, p < 0.05. B , F11 cells, transfected with the pCMV-human SH3BP5 vector, were immunostained with the SH3BP5 antibody ( red ), not preabsorbed ( left ), preabsorbed with GST ( middle ), or preabsorbed with GST-SH3BP5 ( right ), which had been immobilized onto glutathione-Sepharose, for 24 h. Nuclei were stained with DAPI ( blue ). C , immunocytochemical analysis of SH-SY5Y cells with the SH3BP5 antibody ( red ), incubated with 100 n m Humanin G ( HNG ) or distilled water ( DW ) in the presence or the absence of 5 μ m AG490 for 24 h. Nuclei were stained with DAPI ( blue ).

    Techniques Used: Real-time Polymerase Chain Reaction, Incubation, Transfection, Plasmid Preparation, Staining

    14) Product Images from "Structural and Functional Consequences of Tyrosine Phosphorylation in the LRP1 Cytoplasmic Domain"

    Article Title: Structural and Functional Consequences of Tyrosine Phosphorylation in the LRP1 Cytoplasmic Domain

    Journal:

    doi: 10.1074/jbc.M709514200

    Phosphorylation dependence of LRP1-Shp2 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with recombinant His-tagged Shp2 SH2 domains. Bound proteins were visualized by anti-His
    Figure Legend Snippet: Phosphorylation dependence of LRP1-Shp2 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with recombinant His-tagged Shp2 SH2 domains. Bound proteins were visualized by anti-His

    Techniques Used: Incubation, Recombinant

    CD Spectrum of free LRP1-CT. GST-LRP1-CT bound to glutathione-Sepharose was cleaved in solution with thrombin (1:50) in TBS, pH 7.4, 2.5 m m CaCl 2 . The free LRP1-CT was rapidly concentrated by microcolumn centrifugation before analysis. The sample
    Figure Legend Snippet: CD Spectrum of free LRP1-CT. GST-LRP1-CT bound to glutathione-Sepharose was cleaved in solution with thrombin (1:50) in TBS, pH 7.4, 2.5 m m CaCl 2 . The free LRP1-CT was rapidly concentrated by microcolumn centrifugation before analysis. The sample

    Techniques Used: Centrifugation

    Phosphorylation at Tyr 4473 abolishes the LRP1-Snx17 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with lysates of HEK293 cells expressing HA-tagged Snx17. Bound proteins
    Figure Legend Snippet: Phosphorylation at Tyr 4473 abolishes the LRP1-Snx17 interaction. A , unphosphorylated and phosphorylated GST-LRP1-CT fusion proteins immobilized on Sepharose beads were incubated with lysates of HEK293 cells expressing HA-tagged Snx17. Bound proteins

    Techniques Used: Incubation, Expressing, Hemagglutination Assay

    15) Product Images from "A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly"

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    Journal: eLife

    doi: 10.7554/eLife.03473

    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
    Figure Legend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Techniques Used: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

    Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011
    Figure Legend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Techniques Used: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

    RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012
    Figure Legend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Techniques Used: Incubation, SDS Page, Staining, Western Blot

    eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005
    Figure Legend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Techniques Used: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

    Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007
    Figure Legend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Techniques Used: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

    GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010
    Figure Legend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Techniques Used: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

    eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009
    Figure Legend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Techniques Used: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

    16) Product Images from "Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation"

    Article Title: Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-017-0080-6

    Interdependency of SMTNL1 phosphorylation, calcium and tropomyosin on calmodulin-binding. SMTNL1-TMB (200 μg, 7 nmol) was incubated with an equimolar amount of Tpm. The mixture was then incubated with CaM-Sepharose (40 μL; ligand density of ~10–14 μmol/mL) in the presence ( a ; Ca-CaM, 5 mM CaCl 2 ) or absence ( b ; apo-CaM, 1 mM EDTA) of calcium. Some experiments were completed with SMTNL1-TMB that had been previously phosphorylated with PKA. After washing, the retention of SMTNL1-TMB or phosphorylated SMTNL1-TMB was analyzed. The band densities were quantified and binding to CaM-Sepharose expressed as percentage of SMTNL1-TMB recovered under maximal binding conditions (i.e., Ca-CaM in the absence of Tpm). All experiments are n = 3–5 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups (a,b,c: comparison among all Ca-CaM conditions; d,e,f: comparison among all apo-CaM conditions; p
    Figure Legend Snippet: Interdependency of SMTNL1 phosphorylation, calcium and tropomyosin on calmodulin-binding. SMTNL1-TMB (200 μg, 7 nmol) was incubated with an equimolar amount of Tpm. The mixture was then incubated with CaM-Sepharose (40 μL; ligand density of ~10–14 μmol/mL) in the presence ( a ; Ca-CaM, 5 mM CaCl 2 ) or absence ( b ; apo-CaM, 1 mM EDTA) of calcium. Some experiments were completed with SMTNL1-TMB that had been previously phosphorylated with PKA. After washing, the retention of SMTNL1-TMB or phosphorylated SMTNL1-TMB was analyzed. The band densities were quantified and binding to CaM-Sepharose expressed as percentage of SMTNL1-TMB recovered under maximal binding conditions (i.e., Ca-CaM in the absence of Tpm). All experiments are n = 3–5 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups (a,b,c: comparison among all Ca-CaM conditions; d,e,f: comparison among all apo-CaM conditions; p

    Techniques Used: Binding Assay, Incubation, Chick Chorioallantoic Membrane Assay

    SMTNL1 phosphorylation precludes Ca-CaM-binding in favour of Tpm-binding under conditions of high Tpm content. SMTNL1-TMB or phosphorylated SMTNL1-TMB (concentrations as described in Fig. 2 ) was pre-incubated with the indicated molar ratio of purified α/β Tpm dimer. The mixtures were then added to CaM-Sepharose in the absence ( a , c ; apo-CaM, 1 mM EDTA), or presence ( b , d ; Ca-CaM, 5 mM CaCl 2 ) of calcium. After extensive washing, bound SMTNL1-TMB was eluted and detected with Coomassie staining of SDS-PAGE gels. The band density was quantified and binding expressed as percentage of maximum binding within each condition. All experiments are n = 3 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups ( p
    Figure Legend Snippet: SMTNL1 phosphorylation precludes Ca-CaM-binding in favour of Tpm-binding under conditions of high Tpm content. SMTNL1-TMB or phosphorylated SMTNL1-TMB (concentrations as described in Fig. 2 ) was pre-incubated with the indicated molar ratio of purified α/β Tpm dimer. The mixtures were then added to CaM-Sepharose in the absence ( a , c ; apo-CaM, 1 mM EDTA), or presence ( b , d ; Ca-CaM, 5 mM CaCl 2 ) of calcium. After extensive washing, bound SMTNL1-TMB was eluted and detected with Coomassie staining of SDS-PAGE gels. The band density was quantified and binding expressed as percentage of maximum binding within each condition. All experiments are n = 3 and were analyzed by one-way ANOVA with Tukey’s post hoc analysis. Different letters indicate significant differences among groups ( p

    Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Incubation, Purification, Staining, SDS Page

    The phosphorylation of SMTNL1 by PKA alters its tropomyosin-binding potential. a The SMTNL1 protein contains: a C -terminal calponin homology (CH) domain, Ser301 PKA-phosphorylation site, Ca-calmodulin (CaM)-binding domain (CBD1), apo-CaM-binding domain (CBD2), and tropomyosin (Tpm)-binding domain (indicated as the hatched area). b Purified recombinant SMTNL1-TMB was incubated with PKA as described in the Methods section. Samples were withdrawn at the indicated times and subjected to Phos-tag SDS-PAGE; two discrete bands representing unphosphorylated SMTNL1-TMB (0P, TMB) or SMTNL1-TMB phosphorylated at Ser301 (1P, pTMB) were detected by Coomassie stain. Samples subjected to Phos-tag SDS-PAGE in the absence of MnCl 2 confirmed the shift in band migration to be a result of phosphorylation. c Unphosphorylated or phosphorylated SMTNL1-TMB (200 μg) was incubated with 40 μL of Tpm-Sepharose (α/β-heterodimer: Tpm1.4/Tpm2.1). Bound SMTNL1-TMB was eluted with boiling 0.1% SDS solution and detected with Coomassie staining of SDS-PAGE gels. d The SMTNL1-TMB bands were quantified by densitometry, and binding to Tpm-Sepharose was expressed as percentage of the SMTNL1-TMB binding found for the unphosphorylated state. All experiments are n = 3–5 and were analyzed by Student’s t -test. *- Significantly different from unphosphorylated SMTNL1-TMB, p
    Figure Legend Snippet: The phosphorylation of SMTNL1 by PKA alters its tropomyosin-binding potential. a The SMTNL1 protein contains: a C -terminal calponin homology (CH) domain, Ser301 PKA-phosphorylation site, Ca-calmodulin (CaM)-binding domain (CBD1), apo-CaM-binding domain (CBD2), and tropomyosin (Tpm)-binding domain (indicated as the hatched area). b Purified recombinant SMTNL1-TMB was incubated with PKA as described in the Methods section. Samples were withdrawn at the indicated times and subjected to Phos-tag SDS-PAGE; two discrete bands representing unphosphorylated SMTNL1-TMB (0P, TMB) or SMTNL1-TMB phosphorylated at Ser301 (1P, pTMB) were detected by Coomassie stain. Samples subjected to Phos-tag SDS-PAGE in the absence of MnCl 2 confirmed the shift in band migration to be a result of phosphorylation. c Unphosphorylated or phosphorylated SMTNL1-TMB (200 μg) was incubated with 40 μL of Tpm-Sepharose (α/β-heterodimer: Tpm1.4/Tpm2.1). Bound SMTNL1-TMB was eluted with boiling 0.1% SDS solution and detected with Coomassie staining of SDS-PAGE gels. d The SMTNL1-TMB bands were quantified by densitometry, and binding to Tpm-Sepharose was expressed as percentage of the SMTNL1-TMB binding found for the unphosphorylated state. All experiments are n = 3–5 and were analyzed by Student’s t -test. *- Significantly different from unphosphorylated SMTNL1-TMB, p

    Techniques Used: Binding Assay, Chick Chorioallantoic Membrane Assay, Purification, Recombinant, Incubation, SDS Page, Staining, Migration

    17) Product Images from "A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly"

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    Journal: eLife

    doi: 10.7554/eLife.03473

    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
    Figure Legend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Techniques Used: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

    Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011
    Figure Legend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Techniques Used: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

    RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012
    Figure Legend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Techniques Used: Incubation, SDS Page, Staining, Western Blot

    eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005
    Figure Legend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Techniques Used: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

    Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007
    Figure Legend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Techniques Used: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

    GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010
    Figure Legend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Techniques Used: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

    eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009
    Figure Legend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Techniques Used: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

    18) Product Images from "Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9"

    Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025694

    Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.
    Figure Legend Snippet: Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.

    Techniques Used: Silver Staining, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Construct, Staining, Marker

    Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.
    Figure Legend Snippet: Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.

    Techniques Used: Hemagglutination Assay, Sequencing, Affinity Purification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant

    19) Product Images from "Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9"

    Article Title: Exportin 4 Interacts with Sox9 through the HMG Box and Inhibits the DNA Binding of Sox9

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025694

    Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.
    Figure Legend Snippet: Identification of Exp4 as a major interaction partner of Sox9. (A) Silver staining of Sox9 binding proteins separated by NuPAGE. Nuclear extracts prepared from HeLa cells (HeLa NE) were incubated with (lanes 3, 4) or without FLAG-tagged Sox9 (FLAG-Sox9, lanes 1, 2). After recovery with anti-FLAG M2 antibody-conjugated agarose, the proteins were subjected to NuPAGE. The closed arrowhead indicates FLAG-Sox9, and the open arrowhead indicates the protein that was specifically recovered by FLAG-Sox9 (lane 4). (B) Nuclear extracts from U2OS cells were subjected to immunoprecipitation with anti-Sox9 antibody, and the precipitates were subjected to Western blotting analysis using anti-Exp4 antibody (right lane). Normal rabbit IgG was used as a control (middle lane). 1% of the nuclear extract was applied as a control (left lane). (C) The schematic depicts the truncated forms of Sox9 fused with GST (dark gray boxes). The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box (103–181 a.a.). (D) The upper panel shows Western blotting analysis of the protein samples co-precipitated with GST-fused truncated forms of Sox9 using an anti-Exp4 antibody. 5% of the nuclear extract was applied as a control (left lane). Numbers represent the corresponding GST-fused truncated Sox9 constructs shown in C. The lower panel shows CBB staining of NuPAGE for the GST fusion proteins used in this experiment. Numbers on the right represent the molecular weights of the marker proteins.

    Techniques Used: Silver Staining, Binding Assay, Incubation, Immunoprecipitation, Western Blot, Construct, Staining, Marker

    Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.
    Figure Legend Snippet: Interaction of Exp4 with Sox family members. (A) Schematic representation of HA-tagged Sox proteins used in this study. The numbers indicate the amino acid residues. The HMG box domain is shown as a light gray box. The percentage of amino acid identity with the amino acid sequence of the HMG domain of Sox9 is given. (B) The panels show HA-affinity purification of proteins from extracts of HEK293 cells which were transiently transfected with FLAG-Exp4 and HA-Sox9, HA-Sox2, or HA-Sox11. Mock refers to the empty control plasmid. Starting materials (2% input) and bound fractions (IP, immunoprecipitation) were analyzed by NuPAGE and Western blotting. HA-tagged proteins are asterisked in the lower panels. The arrow indicates nonspecific bands. (C) The GST-fused HMG box domains of each Sox protein were separated by NuPAGE and stained with CBB (lower panel). The fusion proteins were incubated with recombinant Exp4 proteins. Proteins bound to glutathione-Sepharose were analyzed by Western blotting with anti-Exp4 antibody (upper panel). 20% input represents the control.

    Techniques Used: Hemagglutination Assay, Sequencing, Affinity Purification, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Recombinant

    20) Product Images from "Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks"

    Article Title: Bora Downregulation Results in Radioresistance by Promoting Repair of Double Strand Breaks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119208

    Bora inhibits MDC1 foci formation via interaction with MDC1 BRCT domain in a phosphorylation-dependent manner. A. Bora interacts with MDC1. IP was performed using anti-Bora antibody followed by blotting with anti-MDC1 antibody in 293T cells. B. Bora interacts with MDC1 via the MDC1 BRCT domain. Lysates from 293T cells overexpressing FLAG-tagged Bora were incubated with GST-BRCT or GST-FHA fusion protein immobilized on the glutathione agarose beads for 2 h before washing. The elution was subsequently analyzed by Western blot with anti-FLAG antibody. C. Effect of phosphorylation on Bora-MDC1 interaction. Lysates from 293T cells overexpressing FLAG-tagged Bora and HA-tagged MDC1 was either incubated with buffer alone or with lambda phosphatase for 15 min at 30°C. The mixture was then incubated with FLAG beads. There was a significant decrease in the binding between Bora and HA-tagged MDC1 in the presence of lambda phosphatase regardless of IR treatment. D. Bora C terminus fragment (313–559 aa), but not N terminus (1–312 aa) co-immunoprecipitates with HA-tagged MDC1. 293T cells were co-transfected with plasmids encoding FLAG-tagged Bora or various deletion constructs, and plasmids encoding HA-tagged MDC1. Lysates were incubated with FLAG beads, followed by Western blot analysis with anti-HA antibody.
    Figure Legend Snippet: Bora inhibits MDC1 foci formation via interaction with MDC1 BRCT domain in a phosphorylation-dependent manner. A. Bora interacts with MDC1. IP was performed using anti-Bora antibody followed by blotting with anti-MDC1 antibody in 293T cells. B. Bora interacts with MDC1 via the MDC1 BRCT domain. Lysates from 293T cells overexpressing FLAG-tagged Bora were incubated with GST-BRCT or GST-FHA fusion protein immobilized on the glutathione agarose beads for 2 h before washing. The elution was subsequently analyzed by Western blot with anti-FLAG antibody. C. Effect of phosphorylation on Bora-MDC1 interaction. Lysates from 293T cells overexpressing FLAG-tagged Bora and HA-tagged MDC1 was either incubated with buffer alone or with lambda phosphatase for 15 min at 30°C. The mixture was then incubated with FLAG beads. There was a significant decrease in the binding between Bora and HA-tagged MDC1 in the presence of lambda phosphatase regardless of IR treatment. D. Bora C terminus fragment (313–559 aa), but not N terminus (1–312 aa) co-immunoprecipitates with HA-tagged MDC1. 293T cells were co-transfected with plasmids encoding FLAG-tagged Bora or various deletion constructs, and plasmids encoding HA-tagged MDC1. Lysates were incubated with FLAG beads, followed by Western blot analysis with anti-HA antibody.

    Techniques Used: Incubation, Western Blot, Hemagglutination Assay, Binding Assay, Transfection, Construct

    Bora S325A phosphorylation mutant causes increased MDC1 and 53BP1 IRIF formation, DNA repair and resistance to IR treatment. A. Bora S325 phosphorylation is required for its association with MDC1. Lysates from 293T cells overexpressing FLAG-tagged WT Bora, the S325E mutant and the S325A mutant with or without IR treatment were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads and subsequent analysis by Western blotting with anti-FLAG antibody. B. Effect of Bora deletion and mutant constructs on MDC1 and 53BP1 IRIF formation. Left Panel: Foci formation. Hela cells were transfected with wild type, Bora deletion, S501A or S325A mutant FLAG-tagged constructs. Forty-eight h after the transfection, cells were treated with 10 Gy IR and immunostained with indicated antibodies. Right Panel: Quantification of MDC1 and 53BP1-IRIF formation per cell is shown after 10 Gy IR. Error bars represent SEM calculated based on100 cells. C. Percentage of GFP positive cells observed in DR-GFP reporter assay in Hela cells that overexpressed different Bora mutant or deletion constructs. Data are presented as mean ± SEM from three independent experiments. Significance was calculated between WT Bora and S325A mutant. D. Effect of Bora deletion and S501mutant constructs on the Bora binding to MDC1 and IR sensitivity. Left Panel: Immunoprecipitation. Lysates from 293T cells overexpressing FLAG-tagged WT Bora and Bora mutants as well as Bora N or C terminal constructs were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads, with subsequent Western blot analysis with anti-FLAG antibody. Right Panel: Colony forming assays. Bora stably knockdown cell lines were transfected with WT Bora, S501A or C and N terminal constructs, and then treated with increasing dose of IR and cytotoxicity was determined by colony forming assays. E. Effect of S325 mutant construct on IR sensitivity. HupT3 and Hela cell lines with Bora stably knockdown were transfected with WT Bora and S325A mutant, and then treated with increasing dose of IR and cytotoxicity was determined by MTS assays and colony forming assays. P-values were calculated for the difference in AUC values between WT and S325A mutant.
    Figure Legend Snippet: Bora S325A phosphorylation mutant causes increased MDC1 and 53BP1 IRIF formation, DNA repair and resistance to IR treatment. A. Bora S325 phosphorylation is required for its association with MDC1. Lysates from 293T cells overexpressing FLAG-tagged WT Bora, the S325E mutant and the S325A mutant with or without IR treatment were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads and subsequent analysis by Western blotting with anti-FLAG antibody. B. Effect of Bora deletion and mutant constructs on MDC1 and 53BP1 IRIF formation. Left Panel: Foci formation. Hela cells were transfected with wild type, Bora deletion, S501A or S325A mutant FLAG-tagged constructs. Forty-eight h after the transfection, cells were treated with 10 Gy IR and immunostained with indicated antibodies. Right Panel: Quantification of MDC1 and 53BP1-IRIF formation per cell is shown after 10 Gy IR. Error bars represent SEM calculated based on100 cells. C. Percentage of GFP positive cells observed in DR-GFP reporter assay in Hela cells that overexpressed different Bora mutant or deletion constructs. Data are presented as mean ± SEM from three independent experiments. Significance was calculated between WT Bora and S325A mutant. D. Effect of Bora deletion and S501mutant constructs on the Bora binding to MDC1 and IR sensitivity. Left Panel: Immunoprecipitation. Lysates from 293T cells overexpressing FLAG-tagged WT Bora and Bora mutants as well as Bora N or C terminal constructs were incubated with GST- MDC1 BRCT fusion protein immobilized on the glutathione agarose beads, with subsequent Western blot analysis with anti-FLAG antibody. Right Panel: Colony forming assays. Bora stably knockdown cell lines were transfected with WT Bora, S501A or C and N terminal constructs, and then treated with increasing dose of IR and cytotoxicity was determined by colony forming assays. E. Effect of S325 mutant construct on IR sensitivity. HupT3 and Hela cell lines with Bora stably knockdown were transfected with WT Bora and S325A mutant, and then treated with increasing dose of IR and cytotoxicity was determined by MTS assays and colony forming assays. P-values were calculated for the difference in AUC values between WT and S325A mutant.

    Techniques Used: Mutagenesis, Incubation, Western Blot, Construct, Transfection, Reporter Assay, Binding Assay, Immunoprecipitation, Stable Transfection

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    Article Snippet: For the GST pull-down assays equal amounts of fusion proteins were incubated with a defined quantity of cell lysate. .. The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Protein expression was then induced with the addition of 0.4 m m isopropyl 1-thio-β-galactopyranoside, and cultures were incubated a further 2.5 h at 30 °C. .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
    Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1. .. Beads were washed twice in PBS and then incubated with 1 mg of THP-1 cell lysate for 3 h at 4°C as described previously ( ).

    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
    Article Snippet: In vitro pull-down experiments : Protein samples were mixed in their correct stoichiometric ratios, using 10 µM She2p wt/ Δhelix E, 10 µM She3p-His6 and 5 µM GST-Myo4-C in a final volume of 100 µl pull-down buffer (20 mM Hepes pH 7.8, 140 mM or 200 mM NaCl, 2 mM MgCl2 , 2 mM DTT). .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker. .. Binding reactions were washed 4 times with 200 µl pull-down buffer and each time spun down at 400 × g, 4 °C for 1 min.

    Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A
    Article Snippet: Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. In in vitro binding assay, recombinant GST-tagged PUM2 (40 µg) on Glutathione-Sepharose beads was incubated with 100 µg purified recombinant His-tagged Aurora-A in binding buffer at 4°C for overnight.

    Article Title:
    Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios. .. Beads were incubated with lysate for 1 h at 4°C, washed three times with lysis buffer, pelleted, and mixed with 100 μl of SDS-sample buffer.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: The cells, which were suspended in 4.8 l LeMaster medium (LeMaster & Richards, 1985 ) supplemented with 25 µg ml−1 SeMet and 100 µg ml−1 ampicillin and grown to an OD of 0.5–0.8, were incubated at 303 K for 4 h after protein expression had been induced with 1 m M IPTG (final concentration). .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Expressing:

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: Paragraph title: Expression and purification of AHSPWT /α-Hb complexes ... The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: For purification of FLAG-tagged proteins from S. pombe cells, cells expressing FLAG-tagged proteins were cultured in YES medium and collected when an optical density of 1.2 at 600 nm was reached. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair
    Article Snippet: Paragraph title: Protein expression and purification ... Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction.

    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
    Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer
    Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of glutathione sepharose beads (GE Healthcare).

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Protein expression was then induced with the addition of 0.4 m m isopropyl 1-thio-β-galactopyranoside, and cultures were incubated a further 2.5 h at 30 °C. .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences).

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Modification:

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Sc787 cells cotransformed with plasmids carrying GST- GAL3 and GAL80 were grown to midlog phase and whole-cell extracts containing GST-Gal3 and Gal80 were prepared as described , using a modified lysis buffer (20 m m HEPES pH 7.4, 0.5% Triton X-100, 200 m m NaCl, 0.5 m m EDTA, 2 m m DTT, and 5 m m MgCl2 ). .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    Western Blot:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. The lysate was clarified by centrifugation (Beckman JA-17 rotor, 15 krpm, 30 min, 4°C) and mixed with Ni-NTA (Qiagen) beads for 1 hour at 4°C.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: Paragraph title: Immunoprecipitation and Western blot analysis ... GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
    Article Snippet: Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1. .. Beads were washed twice in PBS and then incubated with 1 mg of THP-1 cell lysate for 3 h at 4°C as described previously ( ).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The beads were pelleted and washed three times with 500 μl of lysis buffer either with or without ATP and galactose.

    Transformation Assay:

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: Briefly, the plasmid was transformed into BL21 (DE3) cells and grown in Terrific Broth at 37 °C. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Inserts were sequenced to confirm their fidelity, and then the plasmid constructs were transformed into the Epicurian Coli® BL21-CodonPlus™ strain (Stratagene, Cedar Creek, TX). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: Sequences encoding full-length Sec10 or its amino-terminal region (Sec10NTer; aa 378–708) were subcloned into pGEX4T1 at the carboxy terminus of GST and transformed in E. coli BL21(DE3) strain. .. The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences).

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: The resulting plasmid was transformed into Escherichia coli B834(DE3) (Novagen, Madison, Wisconsin, USA). .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Transfection:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were harvested and lysed. .. GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare).

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Concentration Assay:

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: The cells, which were suspended in 4.8 l LeMaster medium (LeMaster & Richards, 1985 ) supplemented with 25 µg ml−1 SeMet and 100 µg ml−1 ampicillin and grown to an OD of 0.5–0.8, were incubated at 303 K for 4 h after protein expression had been induced with 1 m M IPTG (final concentration). .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Protease Inhibitor:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Cells were then lysed with glass beads in lysis buffer A {50 mM Tris-HCl (ph 8.0), 150 mM NaCl, 0.1% NP-40, 10% glycerol, 50 mM NaF, 1 mM Na3 VO4 , 5 mM EDTA, 5 mM N -methylmaleimide, 1 µM microcyctin, 0.1 µM okadaic acid, 0.2 mM p -4-amidoinophenyl-methane sulfonyl fluoride hydrochloride monohydrate (p -APMSF) and Roche complete EDTA-free protease inhibitor cocktail} using a FastPrep cell disrupter (Qbiogene) for two cycles of 20 seconds each at speed 6, with a one-minute interval on ice between the two cycles. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Protease inhibitor cocktails (PIC) (PIC-D, 88 mg/ml PMSF and 1 mg/ml pepstatin A in DMSO; PIC-W, 157 mg/ml benzamidine, 0.5 mg leupeptin, and 0.5 mg bestatin in water) were added to all lysis buffer and all subsequent solutions at 1/1000 dilutions. .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    Cell Culture:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: For purification of FLAG-tagged proteins from S. pombe cells, cells expressing FLAG-tagged proteins were cultured in YES medium and collected when an optical density of 1.2 at 600 nm was reached. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title:
    Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios.

    Hemagglutination Assay:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were harvested and lysed. .. GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare).

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ). .. GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Polymerase Chain Reaction:

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: These PCR products were subcloned in-frame into the bacterial expression vector pGEX-2T (Amersham Biosciences). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Sonication:

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: The bacterial cultures were centrifuged, and the resulting pellets were resuspended in 500 μl of GST-lysis buffer (1 m m EDTA, 0.5% Nonidet P-40) and protease inhibitors in standard phosphate-buffered saline (PBS). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: Cells were harvested by centrifugation, suspended in 1 m M EDTA, 5 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 µ M leupeptin, 1 µ M pepstatin A, 0.1 mg ml−1 lysozyme, Dulbecco’s phosphate-buffered saline that contained neither calcium chloride nor magnesium chloride (PBS; Sigma–Aldrich, St Louis, Missouri, USA) and sonicated and the crude lysate was centrifuged. .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Recombinant:

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
    Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
    Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1. .. Beads were washed twice in PBS and then incubated with 1 mg of THP-1 cell lysate for 3 h at 4°C as described previously ( ).

    Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A
    Article Snippet: pET29a-Aurora-A and pGEX4T2-PUM2 constructs were tramsformed in E. Coli BL21 (DE3). .. Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. In in vitro binding assay, recombinant GST-tagged PUM2 (40 µg) on Glutathione-Sepharose beads was incubated with 100 µg purified recombinant His-tagged Aurora-A in binding buffer at 4°C for overnight.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: Paragraph title: Purification of recombinant proteins expressed in E. coli ... Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP.

    GST Pulldown Assay:

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
    Article Snippet: Paragraph title: GST pulldown assay. ... Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1.

    Pull Down Assay:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: Paragraph title: GST pull-down assay ... GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare).

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: Paragraph title: Protein purification and GST pull-down assay ... Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer
    Article Snippet: Paragraph title: GST pull-down assay ... The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of glutathione sepharose beads (GE Healthcare).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Paragraph title: Pull-down assay for GST-Gal3 and Gal80 interaction: ... Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    Methylation:

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP.

    Mutagenesis:

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: The mutations Thr431Lys, Gln432Leu, Thr451Ala and Ala624Gly were then introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, USA). .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Subcloning:

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    Flow Cytometry:

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C. .. The beads were washed, and the protein was eluted using the lysis buffer supplemented with 250 mM imidazole or 10 mM glutathione.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: Cells were harvested by centrifugation, suspended in 1 m M EDTA, 5 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 µ M leupeptin, 1 µ M pepstatin A, 0.1 mg ml−1 lysozyme, Dulbecco’s phosphate-buffered saline that contained neither calcium chloride nor magnesium chloride (PBS; Sigma–Aldrich, St Louis, Missouri, USA) and sonicated and the crude lysate was centrifuged. .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS. .. Proteins that bound glutathione were eluted in 20 m M Tris–HCl pH 7.9, 10 m M reduced glutathione, 1 m M EDTA, 5 m M 2-mercaptoethanol and then dialyzed against 20 m M Tris–HCl pH 7.9, 1 m M EDTA, 5 m M 2-­mercaptoethanol.

    Purification:

    Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B
    Article Snippet: For IL-6 induction, the cells were serum-starved for at least 16 h, and IL-6 was added at a final concentration of 10 ng/ml for 30 min. .. TFIIS-GST was purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase
    Article Snippet: Immunoblots were visualized by enhanced chemiluminescence method. .. GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: The obtained solution was incubated in the presence of 1% Triton X-100 for 1 h at 4°C. .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The insoluble fraction remaining after solubilization was resuspended in one volume of PBS containing 10% sodium dodecyl sulfate (SDS), sonicated and analyzed by SDS-PAGE and western blotting.

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. E. coli BL21(DE3) cells expressing His6 -Swi3 were suspended in lysis buffer H (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 10% Glycerol, 0.25% Tween 20, 10 mM β-mercaptoethanol, and 1 mM PMSF) containing 10 mM imidazole and lysed by sonication using a Branson Digital Sonifier.

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair
    Article Snippet: Paragraph title: Protein expression and purification ... Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction.

    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
    Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer
    Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of glutathione sepharose beads (GE Healthcare). .. Equal amounts of beads were incubated with 500 μg of LNCaP or AR-transfected COS-1 cell extract in GST buffer [150 m m NaCl, 20 m m Tris (pH 8), 1 m m EDTA and 0.5% (vol/vol) NP-40] overnight at 4 C before being washed five times in the same buffer before resuspension in sodium dodecyl sulfate loading sample buffer, SDS-PAGE, and immunoblotting.

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: The bacterial cultures were centrifuged, and the resulting pellets were resuspended in 500 μl of GST-lysis buffer (1 m m EDTA, 0.5% Nonidet P-40) and protease inhibitors in standard phosphate-buffered saline (PBS). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
    Article Snippet: She2p, She2p (Δhelix E), and She3p were expressed and purified as previously described. .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Protein Purification:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: Paragraph title: Protein purification and GST pull-down assay ... Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair
    Article Snippet: The coding sequences of TDG, Dnmt3a were cloned into pGEX4T-3 (Pharmacia) for expression of the GST-fusions in the E.coli strain BL21 (DE3). .. Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction. .. For GST–TDG, the eluate was dialyzed and loaded again onto a Mono S HR 5/5 cation exchange column (Amersham Biosciences) equilibrated with buffer A [25 mM sodium phosphate (pH 7.0), 0.1 M NaCl, 1 mM EDTA, 10% glycerol, 0.01% Triton X-100, 1 mM DTT and 0.25 mM PMSF].

    Positron Emission Tomography:

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    Immunoprecipitation:

    Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B
    Article Snippet: Paragraph title: GST Pulldown and Immunoprecipitation Assays ... TFIIS-GST was purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: Paragraph title: Immunoprecipitation and Western blot analysis ... GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A
    Article Snippet: Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    Fast Protein Liquid Chromatography:

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The released recombinant proteins were recovered in the supernatant after centrifugation, while GST moiety and Prescission Protease remained bound to the matrix.

    Lysis:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare). .. HEK293T cell lysates or PPARγ2-ubiquitin conjugates were incubated with the beads prebound by GST-tagged proteins for 1 hr at 4°C on a rotating platform.

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: The lysis was completed by brief ultrasonic pulses using a Sonifier II disrupter (Branson Ultrasonic, Carouge-Geneva, Switzerland). .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. E. coli BL21(DE3) cells expressing His6 -Swi3 were suspended in lysis buffer H (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 10% Glycerol, 0.25% Tween 20, 10 mM β-mercaptoethanol, and 1 mM PMSF) containing 10 mM imidazole and lysed by sonication using a Branson Digital Sonifier.

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
    Article Snippet: For the GST pull-down assays equal amounts of fusion proteins were incubated with a defined quantity of cell lysate. .. The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer. .. The protein samples were then analyzed by SDS-PAGE and Western blotting.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    Article Title:
    Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios.

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: The whole-cell extract (∼1 mg) was brought up to a volume of 500 μl with lysis buffer containing 2 m m ATP and 25 m m galactose. .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The whole-cell extracts were then incubated with 50 μl of 50% glutathione Sepharose beads on a rotator at 4° for 2 hr.

    Protein Interaction Assay:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Plasmid Preparation:

    Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase
    Article Snippet: Immunoblots were visualized by enhanced chemiluminescence method. .. GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: Briefly, the plasmid was transformed into BL21 (DE3) cells and grown in Terrific Broth at 37 °C. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer
    Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of glutathione sepharose beads (GE Healthcare).

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Inserts were sequenced to confirm their fidelity, and then the plasmid constructs were transformed into the Epicurian Coli® BL21-CodonPlus™ strain (Stratagene, Cedar Creek, TX). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae
    Article Snippet: The resulting plasmid was transformed into Escherichia coli B834(DE3) (Novagen, Madison, Wisconsin, USA). .. The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Software:

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. The Ni-NTA beads were washed in lysis buffer H containing 20 mM imidazole, and His6 -Swi3 was eluted with lysis buffer H containing 250 mM imidazole and dialyzed against lysis buffer A. Anti-FLAG M2 agarose beads bound to Swi1-FLAG were mixed with His6 -Swi3 in lysis buffer A, incubated by rotation for 1 hour at 4°C, washed three times in lysis buffer A, and analyzed by Western blotting.

    Binding Assay:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare). .. HEK293T cell lysates or PPARγ2-ubiquitin conjugates were incubated with the beads prebound by GST-tagged proteins for 1 hr at 4°C on a rotating platform.

    Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase
    Article Snippet: Paragraph title: In vitro binding assay ... GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences).

    Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A
    Article Snippet: Paragraph title: Preparation of recombinant protein, In vitro binding assay and in Vitro kinase reaction ... Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: Paragraph title: ARF6–Sec10 binding assays ... The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences).

    In Vitro:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: PPARγ2-ubiquitin conjugates were prepared by an in vitro ubiquitination assay. .. GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare).

    Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase
    Article Snippet: Paragraph title: In vitro binding assay ... GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences).

    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
    Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
    Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
    Article Snippet: In vitro pull-down experiments : Protein samples were mixed in their correct stoichiometric ratios, using 10 µM She2p wt/ Δhelix E, 10 µM She3p-His6 and 5 µM GST-Myo4-C in a final volume of 100 µl pull-down buffer (20 mM Hepes pH 7.8, 140 mM or 200 mM NaCl, 2 mM MgCl2 , 2 mM DTT). .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A
    Article Snippet: Paragraph title: Preparation of recombinant protein, In vitro binding assay and in Vitro kinase reaction ... Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    Size-exclusion Chromatography:

    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
    Article Snippet: The protein was further purified using ion exchange and size-exclusion chromatography as previously described. .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    Affinity Chromatography:

    Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B
    Article Snippet: For IL-6 induction, the cells were serum-starved for at least 16 h, and IL-6 was added at a final concentration of 10 ng/ml for 30 min. .. TFIIS-GST was purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule
    Article Snippet: The obtained solution was incubated in the presence of 1% Triton X-100 for 1 h at 4°C. .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The insoluble fraction remaining after solubilization was resuspended in one volume of PBS containing 10% sodium dodecyl sulfate (SDS), sonicated and analyzed by SDS-PAGE and western blotting.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients
    Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    Produced:

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
    Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1.

    FLAG-tag:

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    Staining:

    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
    Article Snippet: The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer. .. The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    Variant Assay:

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
    Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    SDS Page:

    Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ
    Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer (Glutathione Sepharose 4B, GE Healthcare). .. The supernatant was collected as an unbound fraction, and nonspecific binding was removed by washing 5 times with ice-cold lysis buffer.

    Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B
    Article Snippet: TFIIS-GST was purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase
    Article Snippet: GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ). .. Endogenous Jedi-1 was immunoprecipitated using a previously described polyclonal Jedi-1 antibody ( ).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The beads were pelleted and washed three times with 500 μl of lysis buffer either with or without ATP and galactose.

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    GE Healthcare glutathione sepharose
    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione <t>Sepharose</t> and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014
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    The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: The eS26C77W mutant associated with Klippel-Feil syndrome in Diamond-Blackfan anemia patients is impaired in binding importins. ( A ) The DBA linked eS26D33N and eS26C77W mutants are unable to fully rescue the growth defect of eS26-depleted cells. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 mutants were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. Residues mutated in DBA are depicted in Figure 4—figure supplement 3A . ( B ) DBA linked mutations cause strongly reduced eS26 protein levels. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids encoding for eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-eS26 antibodies. Arc1 served as loading control. ( C ) eS26 mutants linked to DBA accumulate 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for eS26 WT and mutant proteins were grown at 37°C to mid-log phase in glucose containing medium. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( D ) Tsr2 interacts with eS26 mutants linked to DBA. Recombinant GST-Tsr2 was immobilized on Glutathione Sepharose and then incubated with E. coli lysates containing eS26a FLAG , eS26D33NFLAG or eS26C77WFLAG lysates for 1 hr at 4°C. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and detected by Coomassie Blue staining. L = input. ( E ) eS26C77W is impaired in binding to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing eS26 FLAG , eS26D33NFLAG or eS26C77WFLAG for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibody. L = input. ( F ) The GFP-eS26D33N fusion protein is efficiently targeted to the nucleus. WT cells expressing GFP-eS26 and GFP-eS26D33N were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 was analyzed by fluorescence microscopy. Scale bar = 5 µm. DOI: http://dx.doi.org/10.7554/eLife.03473.014

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Mutagenesis, Binding Assay, Transformation Assay, Western Blot, Fluorescence In Situ Hybridization, Labeling, Staining, Recombinant, Incubation, SDS Page, Expressing, Fluorescence, Microscopy

    Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: Tsr2 efficiently releases the conserved eS26 from importins. ( A ) Left panel: sequence alignment of eS26 from the indicated organisms done by ClustalO ( Sievers and Higgins, 2014 ; Sievers et al., 2011 ). Conservation at each position is depicted as a gradient from light blue (50% identity) to dark blue (100% identity). Mutated residues linked to DBA are depicted with orange (Asp33) and green (Cys77) dots. Right panel: location of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 clamps the 3′-end of the mature 18S rRNA at the site where the endonuclease Nob1 cleaves the immature 20S pre-rRNA. Inset depicts the 3′-end portion of 18S rRNA (red) in contact with eS26 (blue). The position of amino acids D33 (orange) and C77 (green) that are mutated in DBA or KFS and the coordinated Zn 2+ ion (black) are depicted. ( B ) RanGTP and the 3′-end of 18S rRNA cannot dissociate the Kap123:eS26 complex. GST-Kap123:eS26a FLAG complexes, immobilized on Glutathione Sepharose, were incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2, 3 nM of the 3′-end of 18S rRNA or the combination of RanGTP and the 3′ end of 18S rRNA for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-eS26 antibodies. L = input. GST-tagged importins are indicated with asterisks. ( C ) eS26 stably associates with Tsr2 after its release from Pse1. Immobilized GST-Pse1:eS26 FLAG complex was treated with 1.5 µM His 6 -Tsr2 or buffer alone. The supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in Figure 4E . GST-tagged Pse1 is indicated with an asterisk. ( D ) RanGTP, but not Tsr2 dissociated the Pse1:Slx9 complex in vitro. Pse1:Slx9 complexes were immobilized on Glutathione Sepharose and incubated with buffer alone or with 1.5 µM RanGTP, 1.5 µM Tsr2 or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C and analyzed as in Figure 4C . GST-tagged importins are indicated with asterisks. ( E ) Tsr2 efficiently dissociates importin:eS26 FLAG complexes. GST-Kap104: eS26 FLAG and GST-Pse1:eS26 FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points (1, 2, 4, 8 min). Washing, elution, and visualization were performed as in Figure 4A . GST-tagged importins are indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.011

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Sequencing, Incubation, SDS Page, Staining, Western Blot, Stable Transfection, In Vitro

    RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: RanGTP and Tsr2 do not release eS31, eS8 and uS14 from Kap123. GST-Kap123 and GST alone were immobilized on Glutathione Sepharose and incubated with E. coli lysate containing ∼4 µM eS14 FLAG , eS31 FLAG or eS8 FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C.GST-Kap123:eS14 FLAG , GST-Kap123:eS31 FLAG , GST-Kap123:eS8 FLAG complexes were incubated with either buffer alone or with 1.5 µM RanGTP or 1.5 µM Tsr2 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using α-FLAG-antibodies. L = input. GST-Kap123 is indicated with asterisks. DOI: http://dx.doi.org/10.7554/eLife.03473.012

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Incubation, SDS Page, Staining, Western Blot

    eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: eS26 is required for cytoplasmic processing of immature 20S pre-rRNA to mature 18S rRNA. ( A ) eS26 is essential for viability in yeast. Left panel: WT, rps26aΔ, rps26bΔ and the conditional mutant P GAL1 - RPS26Arps26bΔ were spotted in 10-fold dilutions on galactose and repressive glucose containing media and grown at 30°C for 2–4 days. Right panel: protein levels of eS26 in whole cell extracts of indicated strains were determined by Western analyses using α-eS26 antibodies. Arc1 protein levels served as loading control. ( B ) eS26-depleted cells accumulate immature 20S pre-rRNA in the cytoplasm. P GAL1 - RPS26Arps26bΔ cells transformed with indicated plasmids were grown in glucose containing liquid media at 37°C to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. ( C ) eS26-depleted cells accumulate 80S-like particles. The indicated strains were grown in glucose containing liquid media at 30°C to mid-log phase. Cell extracts were prepared after cycloheximide treatment and subjected to sedimentation centrifugation on 7–50% sucrose density gradients. Polysome profiles were recorded at OD 254nm (top panels). The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. Sucrose gradients were fractionated, the RNA was extracted, separated on a 2% Agarose gel, stained with GelRed (Biotium, middle panels) and subsequently analyzed by Northern blotting using probes against the indicated rRNAs (bottom panels). Exposure times for phosphoimager screens were 20 min for 25S and 18S rRNA, and 3–4 hr for 20S pre-rRNAs. DOI: http://dx.doi.org/10.7554/eLife.03473.005

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Mutagenesis, Western Blot, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Staining, Sedimentation, Centrifugation, Agarose Gel Electrophoresis, Northern Blot

    Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: Tsr2 and eS26 protein levels in the indicated TAP strains and levels of 20S pre-rRNA and 18S rRNA in the indicated TAP purified particles. ( A ) Noc4-, Enp1- and Rio2-TAP purify pre-40S subunits containing immature 20S pre-rRNA whereas Asc1-TAP purifies a 40S subunit containing mature 18S rRNA. 1 µg of RNA isolated from the indicated pre-40S TAP-eluates was separated on a 2% Agarose gel and probed against indicated rRNAs by Northern blotting. 1 µg of total RNA extracted from WT cells was used as a control. ( B ) eS26 does not co-enrich with the earliest 60S pre-ribosome. Noc4-TAP, the earliest pre-ribosomal particle and Ssf1-TAP, the earliest pre-ribosome in the 60S maturation pathway were isolated. The Calmodulin eluates were visualized by Silver staining and by Western analyses using the indicated antibodies. The CBP signal served as loading controls for the TAPs. ( C ) Tsr2 and eS26 protein levels in indicated TAP strains (also used in Figure 3A ) are equal to levels in WT cells. Whole cell extracts (WCE) were prepared from the indicated strains and analyzed by Western analyses using antibodies against Tsr2 and eS26. The protein Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.007

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Purification, Isolation, Agarose Gel Electrophoresis, Northern Blot, Silver Staining, Western Blot

    GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: GFP-eS26 binds to importins and Tsr2 but is not incorporated into pre-ribosomes. ( A ) Location of N- and C-terminus of eS26 within the mature 40S subunit ( Rabl et al., 2011 ). eS26 N-terminus (green) is embedded deeply within the 40S subunit whereas the C-terminus (red) projects away from the body of the 40S subunit. Red letters indicate the 20 C-terminal residues that are not visualized in the structure ( B ) GFP-eS26 is not found in heavier fractions on sucrose gradients. WT lysates and lysates containing GFP-eS26 were subjected to sucrose gradient sedimentation as described in Figure 1D . The peaks for 40S and 60S subunits, 80S ribosomes and polysomes are indicated. The proteins in the gradient were detected by Western analyses using the indicated antibodies. ( C ) GFP-eS26 binds to Kap123, Kap104 and Pse1. Recombinant GST-Kap123, -Kap104, -Pse1 and GST alone were immobilized on Glutathione Sepharose and then incubated with E. coli lysate containing GFP-eS26 for 1 hr at 4°C. Bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining and Western analyses using α-GFP antibody. L = input. ( D ) GFP-eS26 is unable to rescue the lethality of the eS26 deficient strain. The P GAL1 - RPS26Arps26bΔ strain transformed with different plasmids encoding eS26 or GFP-eS26 were spotted in 10-fold dilutions on selective glucose containing plates and grown at indicated temperatures for 3–7 days. ( E ) GFP-eS26 and GFP-eS26D33N levels are strongly reduced in Tsr2-depleted cells. Whole cell extracts (WCE) prepared from WT and Tsr2-depleted cells were assessed by Western analyses using antibodies against the indicated proteins. Arc1 protein levels served as loading control. ( F ) Upper panel: the Zn 2+ -binding domain of eS26 is required for efficient nuclear uptake. WT cells expressing GFP-eS26 truncations were grown in synthetic media at 30°C to mid-log phase and the localization of GFP-eS26 truncations was analyzed by fluorescence microscopy. Scale bar = 5 µm. Lower panel: Schematic for the eS26 truncations used for fluorescence microscopy. ( G ) GFP-eS26C77W protein levels are strongly reduced in (WCE) extracts. Whole cell extracts were prepared from P GAL1 - RPS26Arps26bΔ cells transformed with plasmids encoding for GFP-eS26 WT and mutant proteins. eS26 protein levels were assessed by Western analyses using α-GFP antibodies. Arc1 served as loading control. DOI: http://dx.doi.org/10.7554/eLife.03473.010

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Sedimentation, Western Blot, Recombinant, Incubation, SDS Page, Staining, Transformation Assay, Binding Assay, Expressing, Fluorescence, Microscopy, Mutagenesis

    eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Journal: eLife

    Article Title: A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    doi: 10.7554/eLife.03473

    Figure Lengend Snippet: eS26, but not Tsr2:eS26 or Tsr2, interacts with importins. Recombinant GST tagged importins, immobilized on Glutathione Sepharose, were incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26 or E. coli lysate containing ∼4 µM eS26 FLAG in PBSKMT and competing E. coli lysates for 1 hr at 4°C. After washing, bound proteins were eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by either Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisk, His 6 -Srp1 is indicated with a rectangle. DOI: http://dx.doi.org/10.7554/eLife.03473.009

    Article Snippet: Recombinant GST-Tsr2 was immobilized in PBSKMT on Glutathione Sepharose (GE healthcare), and incubated with E. coli lysates containing recombinant eS26, eS26FLAG , eS26D33NFLAG, eS26C77WFLAG for 1 hr at 4°C.

    Techniques: Recombinant, Incubation, Purification, SDS Page, Staining, Western Blot

    Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: Recombinant GST-Mo-MLV p12 does not associate with mitotic chromatin but is phosphorylated. (A) A representative immunoblot showing subcellular distribution of GST-p12. GST-tagged Mo-MLV p12_WT (lanes 1–3), p12_mut14 (lanes 4–6) and p12+ h CBS (lanes 7–9) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic (lanes 1, 4 and 7), S3-soluble nuclear (lanes 2, 5 and 8) and P3-chromatin pellet (lanes 3, 6 and 9) were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-tagged Mo-MLV p12_WT, p12_mut14 or p12+ h CBS. Cells were stained for p12 (anti-p12, red) and DNA (DAPI, blue). White boxes indicate mitotic cells. (C) Representative silver-stained SDS-PAGE gel (left) and immunoblot (right) of GST-p12 complexes. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 2), p12_mut14 (lane 3) or p12+ h CBS (lane 4), or GST alone (lane 1). 24 h post-transfection, cells were treated with nocodazole overnight to arrest them in mitosis and then lysed. Cell lysates were normalised on total protein concentration and GST-p12 protein complexes were precipitated with glutathione-sepharose beads. Bead eluates were analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-H2A, anti-H2B, anti-H3 or anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Immunoblot showing DNA pull down assays. 293T cells were transiently-transfected with expression constructs for GST alone (top panel), GST-tagged Mo-MLV p12_WT (middle panel), or IN-HA (bottom panel) for ~40 h. DNA interacting proteins were precipitated from normalised cell lysates with cellulose beads coated with double stranded (lane 2) or single-stranded (lane 3) calf thymus DNA, and analysed by immunoblotting with anti-GST, anti-p12, or anti-IN antibodies, respectively. The arrows indicate full-length GST-p12 (~38 kDa) and IN-HA (~49 kDa) bands in the western blots. (E) GST-p12 phosphorylation. Normalised, mitotic cell lysates expressing GST-tagged Mo-MLV p12_WT (lane 3) or p12_S61A (lanes 1 and 2) were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Prior to SDS-PAGE, one p12_S61A sample was treated with alkaline phosphatase (AP) for 1 h at 37°C. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Recombinant, Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Protein Concentration, Silver Staining, Hemagglutination Assay, Western Blot, Incubation, Imaging

    GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I shows increased chromatin association and phosphorylation in mitosis. (A) A representative immunoblot showing subcellular distribution of GST-p12 mutants. GST-tagged GST-p12_M63I (lanes 1–3) or GST-p12+ h CBS (lanes 4–6) were expressed in 293T cells for ~40 h. Cells were then subjected to biochemical fractionation and equivalent amounts of fractions S2-cytosolic, S3-soluble nuclear and P3-chromatin pellet were analysed by SDS-PAGE and immunoblotting with anti-p12, anti-HSP90 (cytosolic marker) and anti-H2B (chromatin marker) antibodies. (B) Representative confocal microscopy images showing GST-p12 localisation in HeLa cells stably transduced with constructs expressing GST-p12_M63I and GST-p12+ h CBS. Cells were stained for p12 (anti-p12, green) and H2B (anti-H2B, red). Blue boxes indicate mitotic cells and red boxes show interphase cells. (C) Representative silver stained gel (top) and immunoblot (bottom) comparing the interaction of GST-p12_M63I and GST-p12+ h CBS with mitotic and interphase chromatin. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT, M63I or GST-p12+ h CBS for ~24 h before being treated overnight with either nocodazole (to arrest in mitosis) or aphidicolin (to block in interphase). GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC and anti-H2B antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (D) Quantitation of H2B pulled-down with GST-p12 from mitotic versus interphase cell lysates. Median H2B band intensities from immunoblots in (C) were measured using a Li-cor Odyssey imaging system. The increase in H2B precipitation from mitotic cell lysates relative to interphase cell lysates are plotted in the bar chart (mean ± SEM, three biological replicates). (E) GST-p12 phosphorylation in mitosis and interphase. Normalised, interphase or mitotic 293T cell lysates expressing GST-tagged Mo-MLV p12_WT, M63I or S61A were incubated with glutathione-sepharose beads. Bound proteins were separated by SDS-PAGE and the gel was sequentially stained with ProQ diamond (PQ, specifically stains phosphorylated proteins) and Sypro ruby (SR, stains all proteins) dyes. Band intensities were measured using a ChemiDoc imaging system and the bar chart shows PQ/SR ratios, plotted as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Fractionation, SDS Page, Marker, Confocal Microscopy, Stable Transfection, Transduction, Construct, Expressing, Staining, Transfection, Blocking Assay, Silver Staining, Quantitation Assay, Western Blot, Imaging, Incubation

    GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12 recapitulates known interactions of the p12 region of Gag. Cellular proteins interacting with GST-p12 were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. (A) Schematic diagram of the SILAC-MS workflow. GST-protein complexes were isolated from normalised mitotic 293T cell lysates using glutathione-sepharose beads, pooled and subjected to LC-MS/MS analysis. (B) Identification of proteins enriched in the heavy-labelled GST-p12_WT (H) sample relative to light-labelled GST (L) sample. Log 2 (H/L) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Isolation, Liquid Chromatography

    GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-p12_M63I interacts with the same chromatin-associated proteins as PFV CBS. Cellular proteins interacting with GST-p12_M63I were identified using SILAC-MS. Two biological repeats (R1 and R2) were performed. GST-p12_M63I and GST-p12_WT were transiently expressed in 293T cells cultured in light (R0/K0) or medium (R6/K4) SILAC media respectively. Cells were treated with nocodazole for mitotic enrichment and then lysed for glutathione-sepharose bead pull-down assays followed by MS. (A) Identification of proteins enriched in the light-labelled GST-p12_M63I (L) sample relative to medium-labelled GST-p12_WT (M) sample. Log 2 (L/M) silac ratios of the set of MS hits (FDR

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Mass Spectrometry, Cell Culture

    GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-tagged Mo-MLV p12_M63I has a higher affinity for chromatin when phosphorylated. (A and B) The effect of kinase inhibitors on p12 phosphorylation (A) and chromatin association (B). 293T cells transiently-expressing GST-p12_M63I were treated overnight with nocodazole, followed by a kinase inhibitor (LiCl, roscovitine (Ros) or kenpaullone (Ken)) for 3.5 h in the presence of both nocodazole and MG132, before lysis. Normalised cell lysates were incubated with glutathione-sepharose beads, bound proteins were separated by SDS-PAGE and gels were analysed either by sequential staining with ProQ diamond (PQ) and Sypro ruby (SR) dyes (A), or by silver-staining and immunoblotting with anti-CLTC and anti-H2B antibodies. PQ/SR ratios (A) and median H2B band intensities (B) are plotted in the bar charts as mean ± SD, of three technical replicates. (C) Mitotic chromatin association of GST-p12_M63I, S61 double mutants. 293T cells transiently-expressing GST-p12_M63I +/- an S61 mutation (S61A, S61D or S61E), were treated overnight with nocodazole and analysed as in (B). (D) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT or M63I, +/- S61 mutations (S61A, S61D or S61E), and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates).

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Expressing, Lysis, Incubation, SDS Page, Staining, Silver Staining, Mutagenesis, Infection, Activity Assay, Reporter Assay

    GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Journal: PLoS Pathogens

    Article Title: Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis

    doi: 10.1371/journal.ppat.1007117

    Figure Lengend Snippet: GST-Mo-MLV p12_M63I and other p12 orthologs associate with mitotic chromatin. (A) Representative silver stained gel (left) and immunoblot (right) showing binding of a panel of GST-p12 mutants to host proteins. 293T cells were transiently-transfected with expression constructs for GST-tagged Mo-MLV p12_WT (lane 1) and a panel of Mo-MLV p12 mutants: M63I (lane 2), G49R/E50K (lane 3), D25A/L-dom (carrying alanine substitutions of the PPPY motif as well as D25A, which disrupts clathrin binding, lane 4), p12 CTD only (lane 5) or GST-p12+ h CBS (positive control, lane 6) for ~24 h before being treated overnight with nocodazole. GST-p12 protein complexes were precipitated from normalised cell lysates with glutathione-sepharose beads and analysed by SDS-PAGE followed by silver-staining or immunoblotting with anti-CLTC, anti-WWP2, anti-H2A, anti-H2B, anti-H3 and anti-H4 antibodies. Bands corresponding to core histones in the silver-stained gel are starred. (B) Infectivity of Mo-MLV VLPs carrying alterations in p12. HeLa cells were challenged with equivalent RT units of LacZ -encoding VLPs carrying Mo-MLV p12_WT, M63I, G49R/E50K or p12+ h CBS +/- Mut14, and infectivity was measured 72 h post-infection by detection of beta-galactosidase activity in a chemiluminescent reporter assay. The data are plotted as percentage of WT VLP infectivity (mean ± SEM of > 3 biological replicates). (C) An alignment of p12 sequences from selected gammaretroviruses. The CTD region is shaded pink. The S61 and M63 residues of Mo-MLV p12 are highlighted in red and equivalent residues at position 63 and 64 are boxed. CTD peptide sequences used in subsequent BLI assays ( Fig 9 ) are in bold. (D and E) Representative silver stained gel (top) and immunoblot (bottom) showing interaction of a panel of GST-tagged p12 orthologues (D) and GST-tagged FeLV_p12 mutants I52M and A53V (E) to chromatin associated proteins. GST-pull down assays were performed as in (A). (E) The amount of histone H2B pulled-down with GST-p12 was quantified for each sample by estimating median band intensity of immunoblots using a Li-cor Odyssey imaging system and plotted in the bar chart as mean ± SD of 3 technical replicates.

    Article Snippet: 0.5 ml aliquots of lysates at 1.5–3 mg/ml were incubated with glutathione-sepharose beads (100 μl/reaction of a 50% slurry) (GE Healthcare) for 3 h at 4°C with end-over-end rotation.

    Techniques: Staining, Binding Assay, Transfection, Expressing, Construct, Positive Control, SDS Page, Silver Staining, Infection, Activity Assay, Reporter Assay, Western Blot, Imaging

    The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: The Residue Cys118 Plays a Key Role in the Autoubiquitination of UCP. (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: In Vitro, Ubiquitin Assay, SDS Page, Mutagenesis

    UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: UCP Forms Polyubiquitin Chains on Specific Lysine Residues in its Substrate. (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: In Vitro, Ubiquitin Assay, Incubation, Mutagenesis, Hemagglutination Assay, Transfection

    Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: Autoubiquitination of UCP Occurs in an E3-independent Manner. (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: Incubation, In Vitro, Mutagenesis

    The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: The UBC Domain of UCP can Forms Polyubiquitin Chains on the N-terminus. (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: Activity Assay, In Vitro, Ubiquitin Assay, Incubation, Transfection, shRNA, Mutagenesis, Hemagglutination Assay, Expressing

    Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: Intermolecular Active Cys118 Residues are Required for Autoubiquitination of UCP. (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: In Vitro, Ubiquitin Assay, Hemagglutination Assay, Mutagenesis, Transfection, Expressing

    The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.

    Journal: PLoS ONE

    Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

    doi: 10.1371/journal.pone.0163710

    Figure Lengend Snippet: The N-terminal and Core Domains of UCP are Critical for Substrate Binding. (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.

    Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

    Techniques: Binding Assay, Incubation, Hemagglutination Assay, Mutagenesis, Transfection, Functional Assay