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    Name:
    Amersham Hybond P 0 2 PVDF x 4m 1 roll PK
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    Structured Review

    GE Healthcare pvdf membranes
    Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using <t>ECL</t> Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on <t>PVDF</t> membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments

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    Images

    1) Product Images from "Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death"

    Article Title: Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.1

    Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on PVDF membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments
    Figure Legend Snippet: Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on PVDF membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments

    Techniques Used: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Expressing, Labeling, Autoradiography

    2) Product Images from "Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa"

    Article Title: Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa

    Journal: Journal of Histochemistry and Cytochemistry

    doi: 10.1369/0022155413520607

    Specificity of the anti-TEKT2BP1 antibody. (A) Recombinant GST-fused proteins were produced in E. coli and separated by SDS-PAGE. Separated proteins were stained with Coomassie brilliant blue (upper panel) or transferred to a PVDF membrane for immunoblotting
    Figure Legend Snippet: Specificity of the anti-TEKT2BP1 antibody. (A) Recombinant GST-fused proteins were produced in E. coli and separated by SDS-PAGE. Separated proteins were stained with Coomassie brilliant blue (upper panel) or transferred to a PVDF membrane for immunoblotting

    Techniques Used: Recombinant, Produced, SDS Page, Staining

    3) Product Images from "Engineered Conformation-dependent VEGF Peptide Mimics Are Effective in Inhibiting VEGF Signaling Pathways"

    Article Title: Engineered Conformation-dependent VEGF Peptide Mimics Are Effective in Inhibiting VEGF Signaling Pathways

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.216812

    Inhibition of phosphorylation in HUVEC. Cells were grown in 6-well plates and incubated with inhibitor as indicated for 30 min prior to stimulation with rhVEGF (10 ng/ml). Representative Western blots of cell lysates were dissolved in SDS-PAGE, transferred to PVDF membranes, and detected in phosphorylated KDR ( A ) and MAPK p44 ERK1 and p42 ERK2 ( B ) using specific anti-phospho- and anti-total KDR and anti-p44/42 MAPK and anti-CD31, respectively.
    Figure Legend Snippet: Inhibition of phosphorylation in HUVEC. Cells were grown in 6-well plates and incubated with inhibitor as indicated for 30 min prior to stimulation with rhVEGF (10 ng/ml). Representative Western blots of cell lysates were dissolved in SDS-PAGE, transferred to PVDF membranes, and detected in phosphorylated KDR ( A ) and MAPK p44 ERK1 and p42 ERK2 ( B ) using specific anti-phospho- and anti-total KDR and anti-p44/42 MAPK and anti-CD31, respectively.

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

    Inhibition of KDR phosphorylation in 293/KDR cell line. Cells were grown in 6-well plates and incubated with inhibitor as indicated 30 min prior to stimulation with rhVEGF (10 ng/ml). A , representative Western blot using 293/KDR cell lysates that were solved in SDS-PAGE, transferred to PVDF membranes, and probed using specific anti-phospho- and anti-total KDR. B , percentage of VEGFR-2 phosphorylation calculated using human phospho-VEGF R2/KDR DuoSet IC kit (R D Systems).
    Figure Legend Snippet: Inhibition of KDR phosphorylation in 293/KDR cell line. Cells were grown in 6-well plates and incubated with inhibitor as indicated 30 min prior to stimulation with rhVEGF (10 ng/ml). A , representative Western blot using 293/KDR cell lysates that were solved in SDS-PAGE, transferred to PVDF membranes, and probed using specific anti-phospho- and anti-total KDR. B , percentage of VEGFR-2 phosphorylation calculated using human phospho-VEGF R2/KDR DuoSet IC kit (R D Systems).

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

    4) Product Images from "Expression of t-DARPP Mediates Trastuzumab Resistance in Breast Cancer Cells"

    Article Title: Expression of t-DARPP Mediates Trastuzumab Resistance in Breast Cancer Cells

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-08-0121

    t-DARPP associates with ERBB2 and HSP90 proteins in trastuzumab-resistant cells. Immunoprecipitated proteins with t-DARPP, HSP90, or ERBB2 antibodies and the cell lysate ( Input ) from HR-5 cells were resolved on 10% SDS-PAGE and transferred onto Hybond-P polyvinylidene difluoride membranes for Western blot analysis of t-DARPP, HSP90, and ERBB2 proteins. Protein supernatants collected after the first spin of agarose beads were used as controls. Both HSP90 and ERBB2 coprecipitated with t-DARPP.
    Figure Legend Snippet: t-DARPP associates with ERBB2 and HSP90 proteins in trastuzumab-resistant cells. Immunoprecipitated proteins with t-DARPP, HSP90, or ERBB2 antibodies and the cell lysate ( Input ) from HR-5 cells were resolved on 10% SDS-PAGE and transferred onto Hybond-P polyvinylidene difluoride membranes for Western blot analysis of t-DARPP, HSP90, and ERBB2 proteins. Protein supernatants collected after the first spin of agarose beads were used as controls. Both HSP90 and ERBB2 coprecipitated with t-DARPP.

    Techniques Used: Immunoprecipitation, SDS Page, Western Blot

    5) Product Images from "Enhanced archaeal laccase production in recombinant Escherichia coli by modification of N-terminal propeptide and twin arginine translocation motifs"

    Article Title: Enhanced archaeal laccase production in recombinant Escherichia coli by modification of N-terminal propeptide and twin arginine translocation motifs

    Journal: Journal of industrial microbiology & biotechnology

    doi: 10.1007/s10295-012-1152-7

    Electrophoretic comparison of LccA purified from H. volcanii and recombinant E. coli strains. LccA protein from H. volcanii US02, E. coli US04 ( lccAΔtat ) MonoQ (MQ) and gel filtration (GF) fractions, and E. coli US05 ( lccAΔpro ) are indicated.
    Figure Legend Snippet: Electrophoretic comparison of LccA purified from H. volcanii and recombinant E. coli strains. LccA protein from H. volcanii US02, E. coli US04 ( lccAΔtat ) MonoQ (MQ) and gel filtration (GF) fractions, and E. coli US05 ( lccAΔpro ) are indicated.

    Techniques Used: Purification, Recombinant, Filtration

    6) Product Images from "Secondary Metabolism and Development Is Mediated by LlmF Control of VeA Subcellular Localization in Aspergillus nidulans"

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

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003193

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

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

    7) Product Images from "aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity"

    Article Title: aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.07.007

    Segregation of Anterior PAR Proteins Involves Cortical Clusters (A) Representative cortical images of PAR-3, PKC-3, CDC-42, and PH-PLCΔ1 in late-establishment and maintenance-phase zygotes along with zoom of inset region (yellow box). (B) Time-averaged cortical images spanning 180 s reveal anterior-directed tracks of cortical clusters of PAR-3, PAR-6, and PKC-3. Insets highlight the motion (arrows) of a representative single cluster in the image above. (C) Cluster index for the full dataset in (A) and PAR-6::GFP (images not shown). Significance between establishment and maintenance: p
    Figure Legend Snippet: Segregation of Anterior PAR Proteins Involves Cortical Clusters (A) Representative cortical images of PAR-3, PKC-3, CDC-42, and PH-PLCΔ1 in late-establishment and maintenance-phase zygotes along with zoom of inset region (yellow box). (B) Time-averaged cortical images spanning 180 s reveal anterior-directed tracks of cortical clusters of PAR-3, PAR-6, and PKC-3. Insets highlight the motion (arrows) of a representative single cluster in the image above. (C) Cluster index for the full dataset in (A) and PAR-6::GFP (images not shown). Significance between establishment and maintenance: p

    Techniques Used:

    Regulation of PKC-3/PAR-6 Cluster Association by PAR-3/CDC-42 Balance Tunes Responsiveness to Cortical Flows (A) Representative cortical images of PAR-6::GFP at late-establishment and maintenance-phase embryos for indicated conditions, shown along with a zoom of inset region (white boxes). (B) Cluster index measurements of full dataset reveal a gradient of cluster association across conditions. Note that clustering decreases when embryos enter maintenance phase, except for CRT90/ par-3 ( RNAi ) embryos, which show minimal clustering even in establishment phase. (C) ASI measurements of midsection images taken at late-establishment phase for a similar set of embryos as in (A) and (B), but expressing GFP::PKC-3, show a similar trend. (D) Profiles of membrane signal for zygotes in (C) showing average (solid line) and full range of data (shaded) reveal shift of the PKC-3 domain boundary (arrows) toward the anterior in cdc-42 ( RNAi ) embryos and toward the posterior in CRT90-treated zygotes, resulting in significantly (p
    Figure Legend Snippet: Regulation of PKC-3/PAR-6 Cluster Association by PAR-3/CDC-42 Balance Tunes Responsiveness to Cortical Flows (A) Representative cortical images of PAR-6::GFP at late-establishment and maintenance-phase embryos for indicated conditions, shown along with a zoom of inset region (white boxes). (B) Cluster index measurements of full dataset reveal a gradient of cluster association across conditions. Note that clustering decreases when embryos enter maintenance phase, except for CRT90/ par-3 ( RNAi ) embryos, which show minimal clustering even in establishment phase. (C) ASI measurements of midsection images taken at late-establishment phase for a similar set of embryos as in (A) and (B), but expressing GFP::PKC-3, show a similar trend. (D) Profiles of membrane signal for zygotes in (C) showing average (solid line) and full range of data (shaded) reveal shift of the PKC-3 domain boundary (arrows) toward the anterior in cdc-42 ( RNAi ) embryos and toward the posterior in CRT90-treated zygotes, resulting in significantly (p

    Techniques Used: Expressing

    Membrane Localization of PAR-6/PKC-3 Is Decoupled from PAR-3 when PKC-3 Is Inactive (A–F) Representative midsection confocal images of live and fixed zygotes at establishment and maintenance phase comparing control (DMSO, wild-type), pkc-3 ( RNAi ), and PKC-3-inhibited (CRT90, pkc-3 ( ts )) conditions. PAR-6 (A and B) and PKC-3 (C and D) show loss of asymmetric membrane staining in PKC-3-inhibited zygotes at both establishment and maintenance phase (posterior localization indicated by white arrowheads). In pkc-3 ( RNAi ), PAR-6 is absent from the membrane at all times. PAR-3 (E and F) still polarizes in PKC-3-inhibited zygotes, but becomes weaker and less asymmetric during maintenance phase. Note that (B), (D), and (F) show the same wild-type and TS zygotes with the PAR-3 boundary position in TS indicated (red arrowheads) to allow comparison: PAR-6 and PKC-3 are clearly visible at the posterior membrane (white arrowheads), while PAR-3 is undetectable, as in wild-type. Bright foci in (D) are non-specific centrosome staining. (G and H) Normalized ASI measurements for late establishment phase datasets represented in (A) to (F). ASI is normalized to control (wild-type [WT] or DMSO) for each protein. (I and J) Anterior to posterior membrane distributions of PAR-3 (red) and PKC-3 (black) in wild-type (I) and pkc-3 ( ts ) (J) embryos. Arrows highlight the posterior extension of PKC-3 relative to PAR-3. Mean ± SD is shown. (K) Close-up view of the boundary region showing PAR-3 (top) and PKC-3 (bottom) for one representative zygote for wild-type (WT) and pkc-3 ( ts ) backgrounds as indicated. Dashed rectangular selection denotes regions where PKC-3 is present in absence of PAR-3. (L) PKC-3 to PAR-3 ASI ratio for wild-type (WT) and pkc-3 ( ts ). (M) Dual labeling of PAR-2 and PKC-3 in live, CRT90-treated zygotes (top) and fixed, pkc-3 ( ts ) embryos (bottom) reveal overlap of aPAR and pPAR proteins. ∗∗ p
    Figure Legend Snippet: Membrane Localization of PAR-6/PKC-3 Is Decoupled from PAR-3 when PKC-3 Is Inactive (A–F) Representative midsection confocal images of live and fixed zygotes at establishment and maintenance phase comparing control (DMSO, wild-type), pkc-3 ( RNAi ), and PKC-3-inhibited (CRT90, pkc-3 ( ts )) conditions. PAR-6 (A and B) and PKC-3 (C and D) show loss of asymmetric membrane staining in PKC-3-inhibited zygotes at both establishment and maintenance phase (posterior localization indicated by white arrowheads). In pkc-3 ( RNAi ), PAR-6 is absent from the membrane at all times. PAR-3 (E and F) still polarizes in PKC-3-inhibited zygotes, but becomes weaker and less asymmetric during maintenance phase. Note that (B), (D), and (F) show the same wild-type and TS zygotes with the PAR-3 boundary position in TS indicated (red arrowheads) to allow comparison: PAR-6 and PKC-3 are clearly visible at the posterior membrane (white arrowheads), while PAR-3 is undetectable, as in wild-type. Bright foci in (D) are non-specific centrosome staining. (G and H) Normalized ASI measurements for late establishment phase datasets represented in (A) to (F). ASI is normalized to control (wild-type [WT] or DMSO) for each protein. (I and J) Anterior to posterior membrane distributions of PAR-3 (red) and PKC-3 (black) in wild-type (I) and pkc-3 ( ts ) (J) embryos. Arrows highlight the posterior extension of PKC-3 relative to PAR-3. Mean ± SD is shown. (K) Close-up view of the boundary region showing PAR-3 (top) and PKC-3 (bottom) for one representative zygote for wild-type (WT) and pkc-3 ( ts ) backgrounds as indicated. Dashed rectangular selection denotes regions where PKC-3 is present in absence of PAR-3. (L) PKC-3 to PAR-3 ASI ratio for wild-type (WT) and pkc-3 ( ts ). (M) Dual labeling of PAR-2 and PKC-3 in live, CRT90-treated zygotes (top) and fixed, pkc-3 ( ts ) embryos (bottom) reveal overlap of aPAR and pPAR proteins. ∗∗ p

    Techniques Used: Staining, Selection, Labeling

    Polarization through Coupling of PAR-3- and CDC-42-Dependent aPAR Assemblies (i) PKC-3 kinase activity ensures that PKC-3 loads via a PAR-3 intermediate, in which PKC-3 activity is suppressed. This dependence on PAR-3 can be bypassed upon inhibition of PKC-3 (dashed arrow). (ii) Clustering of membrane-associated PAR-3 allows it to be segregated by cortical flow into the anterior, carrying along associated PKC-3 molecules and generating asymmetric sites for further PKC-3 loading. (iii) PKC-3 activation requires conversion into a CDC-42-associated assembly, which relieves inhibition of PKC-3 by PAR-3. (iv) The CDC-42-dependent module is freely diffusible on the membrane and locally excludes pPARs. (v) Dissociation of CDC-42-dependent assemblies limits the spread of active PKC-3 at the membrane from the PAR-3 recruiting site. (vi) PKC-3 returns to the cytoplasm where it must load again via PAR-3.
    Figure Legend Snippet: Polarization through Coupling of PAR-3- and CDC-42-Dependent aPAR Assemblies (i) PKC-3 kinase activity ensures that PKC-3 loads via a PAR-3 intermediate, in which PKC-3 activity is suppressed. This dependence on PAR-3 can be bypassed upon inhibition of PKC-3 (dashed arrow). (ii) Clustering of membrane-associated PAR-3 allows it to be segregated by cortical flow into the anterior, carrying along associated PKC-3 molecules and generating asymmetric sites for further PKC-3 loading. (iii) PKC-3 activation requires conversion into a CDC-42-associated assembly, which relieves inhibition of PKC-3 by PAR-3. (iv) The CDC-42-dependent module is freely diffusible on the membrane and locally excludes pPARs. (v) Dissociation of CDC-42-dependent assemblies limits the spread of active PKC-3 at the membrane from the PAR-3 recruiting site. (vi) PKC-3 returns to the cytoplasm where it must load again via PAR-3.

    Techniques Used: Activity Assay, Inhibition, Flow Cytometry, Activation Assay

    PKC-3 Inhibition Promotes PAR-3-Independent Formation of CDC-42-Dependent PAR-6/PKC-3 Assemblies (A–C) Representative midsection confocal images of live embryos at maintenance phase showing GFP::PKC-3 (A) or PAR-6::GFP (B and C) of DMSO, CRT90-treated, and pkc-3 ( ts ) zygotes subject to RNAi as indicated. (D) Quantification of rescue for datasets represented in (A) to (C), normalized to membrane signal in control RNAi and CRT90-treated/ pkc-3 ( ts ) zygotes for each dataset. (E) Representative midsection confocal images of wild-type and pkc-3 ( ts ) zygotes during polarity establishment subject to RNAi as indicated and immunostained for PKC-3. (F) Quantification of rescue as measured by anterior domain cortical intensity of PKC-3 for datasets represented in (E). For each zygote, anterior PKC-3 cortical intensity is divided by cytoplasmic intensity. Values greater than 1 indicate presence at the membrane. Mean ± 95% confidence interval (CI) (N) is shown. See STAR Methods for further details. (G) Representative midsection confocal images during polarity establishment of wild-type and pkc-3 ( ts ) embryos upon cgef-1 ( RNAi ), stained for PKC-3. Scatterplot representing the anterior domain cortical intensity of PKC-3 as in (F) in cgef-1 ( RNAi ) and pkc-3 ( ts ); cgef-1 ( RNAi ). Mean ± 95% CI (N) is shown. ∗∗ p
    Figure Legend Snippet: PKC-3 Inhibition Promotes PAR-3-Independent Formation of CDC-42-Dependent PAR-6/PKC-3 Assemblies (A–C) Representative midsection confocal images of live embryos at maintenance phase showing GFP::PKC-3 (A) or PAR-6::GFP (B and C) of DMSO, CRT90-treated, and pkc-3 ( ts ) zygotes subject to RNAi as indicated. (D) Quantification of rescue for datasets represented in (A) to (C), normalized to membrane signal in control RNAi and CRT90-treated/ pkc-3 ( ts ) zygotes for each dataset. (E) Representative midsection confocal images of wild-type and pkc-3 ( ts ) zygotes during polarity establishment subject to RNAi as indicated and immunostained for PKC-3. (F) Quantification of rescue as measured by anterior domain cortical intensity of PKC-3 for datasets represented in (E). For each zygote, anterior PKC-3 cortical intensity is divided by cytoplasmic intensity. Values greater than 1 indicate presence at the membrane. Mean ± 95% confidence interval (CI) (N) is shown. See STAR Methods for further details. (G) Representative midsection confocal images during polarity establishment of wild-type and pkc-3 ( ts ) embryos upon cgef-1 ( RNAi ), stained for PKC-3. Scatterplot representing the anterior domain cortical intensity of PKC-3 as in (F) in cgef-1 ( RNAi ) and pkc-3 ( ts ); cgef-1 ( RNAi ). Mean ± 95% CI (N) is shown. ∗∗ p

    Techniques Used: Inhibition, Staining

    PKC-3 Kinase Inhibition Leads to Symmetric Division and Loss of Asymmetry of Downstream Polarity Markers (A–C) Model for symmetry breaking by the PAR system in C. elegans . aPARs (red) initially occupy the membrane and pPARs (blue) are cytoplasmic (A, Meiosis II). A cue (purple) from the centrosome pair (black spheres) segregates aPARs into the anterior and promotes formation of a posterior PAR domain at the opposite pole (A, Establishment). PAR domains are then stable until cytokinesis (A, Maintenance) and drive polarization of cytoplasmic factors such as MEX-5/6 (green) and P granules (orange), which ensure the daughter cells acquire distinct fates (A, Two-cell). (B) Symmetry breaking can occur in two ways: (i) segregation of aPARs by cortical actomyosin flow (advection); and (ii) posterior PAR-2 loading. (C) A complex network of physical and regulatory interactions links the PAR proteins. Membrane binding (gray lines), physical interactions (black lines), as well as positive (→) and negative (⊥) feedback, are shown. Where links are indirect or unknown, dashed lines are used. Both CDC-42 and PAR-3 are required for stable membrane association of PAR-6/PKC-3. PAR-6 and PKC-3 depend on each other for membrane association. PAR-2, LGL-1, and presumably CHIN-1, are able to load onto the membrane independently. PAR-1 also binds membrane but requires PAR-2 to reach maximal concentrations. PKC-3 phosphorylates PAR-1, PAR-2, and LGL-1 and displaces them from the membrane. Exclusion of CHIN-1 from the anterior is dependent on PKC-3, but whether it is a direct target of PKC-3 is unknown. Together, PAR-1, via phosphorylation of PAR-3, and CHIN-1, by suppressing activated CDC-42, prevent invasion of the posterior domain by aPARs. PAR-3 and PAR-2 have been proposed to undergo oligomerization, which is thought to enhance their membrane association (noted by circular arrows). See recent reviews ( Goehring, 2014 , Hoege and Hyman, 2013 , Motegi and Seydoux, 2013 ) for more information. (D) Midsection confocal images of fixed zygotes stained for PAR-2 at polarity maintenance and two-cell stage comparing wild-type, pkc-3 ( ts ), and pkc-3 ( RNAi ) conditions. (E) Midsection fluorescent images of mCherry:PAR-2-expressing zygotes at maintenance and two-cell stage in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). (F) Midsection (PAR-1, LGL-1, PIE-1, MEX-5) or cortical (CHIN-1) fluorescent images of maintenance-phase zygotes expressing markers to various downstream polarity markers in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). Asymmetry in (D) to (F) is quantified by the asymmetry index, with one being normal asymmetry and zero, no asymmetry (ASI, normalized to DMSO/WT controls). ∗ p
    Figure Legend Snippet: PKC-3 Kinase Inhibition Leads to Symmetric Division and Loss of Asymmetry of Downstream Polarity Markers (A–C) Model for symmetry breaking by the PAR system in C. elegans . aPARs (red) initially occupy the membrane and pPARs (blue) are cytoplasmic (A, Meiosis II). A cue (purple) from the centrosome pair (black spheres) segregates aPARs into the anterior and promotes formation of a posterior PAR domain at the opposite pole (A, Establishment). PAR domains are then stable until cytokinesis (A, Maintenance) and drive polarization of cytoplasmic factors such as MEX-5/6 (green) and P granules (orange), which ensure the daughter cells acquire distinct fates (A, Two-cell). (B) Symmetry breaking can occur in two ways: (i) segregation of aPARs by cortical actomyosin flow (advection); and (ii) posterior PAR-2 loading. (C) A complex network of physical and regulatory interactions links the PAR proteins. Membrane binding (gray lines), physical interactions (black lines), as well as positive (→) and negative (⊥) feedback, are shown. Where links are indirect or unknown, dashed lines are used. Both CDC-42 and PAR-3 are required for stable membrane association of PAR-6/PKC-3. PAR-6 and PKC-3 depend on each other for membrane association. PAR-2, LGL-1, and presumably CHIN-1, are able to load onto the membrane independently. PAR-1 also binds membrane but requires PAR-2 to reach maximal concentrations. PKC-3 phosphorylates PAR-1, PAR-2, and LGL-1 and displaces them from the membrane. Exclusion of CHIN-1 from the anterior is dependent on PKC-3, but whether it is a direct target of PKC-3 is unknown. Together, PAR-1, via phosphorylation of PAR-3, and CHIN-1, by suppressing activated CDC-42, prevent invasion of the posterior domain by aPARs. PAR-3 and PAR-2 have been proposed to undergo oligomerization, which is thought to enhance their membrane association (noted by circular arrows). See recent reviews ( Goehring, 2014 , Hoege and Hyman, 2013 , Motegi and Seydoux, 2013 ) for more information. (D) Midsection confocal images of fixed zygotes stained for PAR-2 at polarity maintenance and two-cell stage comparing wild-type, pkc-3 ( ts ), and pkc-3 ( RNAi ) conditions. (E) Midsection fluorescent images of mCherry:PAR-2-expressing zygotes at maintenance and two-cell stage in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). (F) Midsection (PAR-1, LGL-1, PIE-1, MEX-5) or cortical (CHIN-1) fluorescent images of maintenance-phase zygotes expressing markers to various downstream polarity markers in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). Asymmetry in (D) to (F) is quantified by the asymmetry index, with one being normal asymmetry and zero, no asymmetry (ASI, normalized to DMSO/WT controls). ∗ p

    Techniques Used: Inhibition, Flow Cytometry, Binding Assay, Staining, Expressing

    PAR-3 and CDC-42 Have Opposing Regulatory Roles in an In Vivo PKC-3 Activity Assay (A) C1B targeting strategy for inducing PKC-3 membrane loading by PMA. PKC-3 kinase activity is monitored by following loss of PAR-2 from the membrane. (B) Zygotes expressing GFP::C1B alone (GFP::C1B-Ø) or GFP::C1B-PKC-3 along with mCherry::PAR-2 were subject to the indicated treatment. Note that uniform membrane targeting of C1B-PKC-3 leads to reduction of PAR-2 domain size, whereas omitting PMA or expressing C1B alone has no effect. Right: cartoon representation of results. (C) Quantification of PAR-2 domain size ratio for embryos shown in (B). (D) PAR-2 retention in GFP::C1B-PKC-3 expressing zygotes treated with PMA and CRT90 confirms that induced PAR-2 loss is dependent on PKC-3 kinase activity. (E) Zygotes expressing mCherry::PAR-2 with GFP::C1B-Ø or GFP::C1B-PKC-3 subject to par-6 , cdc-42 , or par-3 ( RNAi ) before and 5 min after PMA addition. (F) Quantification of PAR-2 cortex retention comparing GFP::C1B-PKC-3 and GFP::C1B-Ø zygotes after treatment with PMA as in (E). Representative midsection confocal images are shown in (B), (D), and (E) before and 5 min after PMA/DMSO addition. ∗∗∗ p
    Figure Legend Snippet: PAR-3 and CDC-42 Have Opposing Regulatory Roles in an In Vivo PKC-3 Activity Assay (A) C1B targeting strategy for inducing PKC-3 membrane loading by PMA. PKC-3 kinase activity is monitored by following loss of PAR-2 from the membrane. (B) Zygotes expressing GFP::C1B alone (GFP::C1B-Ø) or GFP::C1B-PKC-3 along with mCherry::PAR-2 were subject to the indicated treatment. Note that uniform membrane targeting of C1B-PKC-3 leads to reduction of PAR-2 domain size, whereas omitting PMA or expressing C1B alone has no effect. Right: cartoon representation of results. (C) Quantification of PAR-2 domain size ratio for embryos shown in (B). (D) PAR-2 retention in GFP::C1B-PKC-3 expressing zygotes treated with PMA and CRT90 confirms that induced PAR-2 loss is dependent on PKC-3 kinase activity. (E) Zygotes expressing mCherry::PAR-2 with GFP::C1B-Ø or GFP::C1B-PKC-3 subject to par-6 , cdc-42 , or par-3 ( RNAi ) before and 5 min after PMA addition. (F) Quantification of PAR-2 cortex retention comparing GFP::C1B-PKC-3 and GFP::C1B-Ø zygotes after treatment with PMA as in (E). Representative midsection confocal images are shown in (B), (D), and (E) before and 5 min after PMA/DMSO addition. ∗∗∗ p

    Techniques Used: In Vivo, Activity Assay, Expressing

    8) Product Images from "aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity"

    Article Title: aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.07.007

    Segregation of Anterior PAR Proteins Involves Cortical Clusters (A) Representative cortical images of PAR-3, PKC-3, CDC-42, and PH-PLCΔ1 in late-establishment and maintenance-phase zygotes along with zoom of inset region (yellow box). (B) Time-averaged cortical images spanning 180 s reveal anterior-directed tracks of cortical clusters of PAR-3, PAR-6, and PKC-3. Insets highlight the motion (arrows) of a representative single cluster in the image above. (C) Cluster index for the full dataset in (A) and PAR-6::GFP (images not shown). Significance between establishment and maintenance: p
    Figure Legend Snippet: Segregation of Anterior PAR Proteins Involves Cortical Clusters (A) Representative cortical images of PAR-3, PKC-3, CDC-42, and PH-PLCΔ1 in late-establishment and maintenance-phase zygotes along with zoom of inset region (yellow box). (B) Time-averaged cortical images spanning 180 s reveal anterior-directed tracks of cortical clusters of PAR-3, PAR-6, and PKC-3. Insets highlight the motion (arrows) of a representative single cluster in the image above. (C) Cluster index for the full dataset in (A) and PAR-6::GFP (images not shown). Significance between establishment and maintenance: p

    Techniques Used:

    Regulation of PKC-3/PAR-6 Cluster Association by PAR-3/CDC-42 Balance Tunes Responsiveness to Cortical Flows (A) Representative cortical images of PAR-6::GFP at late-establishment and maintenance-phase embryos for indicated conditions, shown along with a zoom of inset region (white boxes). (B) Cluster index measurements of full dataset reveal a gradient of cluster association across conditions. Note that clustering decreases when embryos enter maintenance phase, except for CRT90/ par-3 ( RNAi ) embryos, which show minimal clustering even in establishment phase. (C) ASI measurements of midsection images taken at late-establishment phase for a similar set of embryos as in (A) and (B), but expressing GFP::PKC-3, show a similar trend. (D) Profiles of membrane signal for zygotes in (C) showing average (solid line) and full range of data (shaded) reveal shift of the PKC-3 domain boundary (arrows) toward the anterior in cdc-42 ( RNAi ) embryos and toward the posterior in CRT90-treated zygotes, resulting in significantly (p
    Figure Legend Snippet: Regulation of PKC-3/PAR-6 Cluster Association by PAR-3/CDC-42 Balance Tunes Responsiveness to Cortical Flows (A) Representative cortical images of PAR-6::GFP at late-establishment and maintenance-phase embryos for indicated conditions, shown along with a zoom of inset region (white boxes). (B) Cluster index measurements of full dataset reveal a gradient of cluster association across conditions. Note that clustering decreases when embryos enter maintenance phase, except for CRT90/ par-3 ( RNAi ) embryos, which show minimal clustering even in establishment phase. (C) ASI measurements of midsection images taken at late-establishment phase for a similar set of embryos as in (A) and (B), but expressing GFP::PKC-3, show a similar trend. (D) Profiles of membrane signal for zygotes in (C) showing average (solid line) and full range of data (shaded) reveal shift of the PKC-3 domain boundary (arrows) toward the anterior in cdc-42 ( RNAi ) embryos and toward the posterior in CRT90-treated zygotes, resulting in significantly (p

    Techniques Used: Expressing

    Membrane Localization of PAR-6/PKC-3 Is Decoupled from PAR-3 when PKC-3 Is Inactive (A–F) Representative midsection confocal images of live and fixed zygotes at establishment and maintenance phase comparing control (DMSO, wild-type), pkc-3 ( RNAi ), and PKC-3-inhibited (CRT90, pkc-3 ( ts )) conditions. PAR-6 (A and B) and PKC-3 (C and D) show loss of asymmetric membrane staining in PKC-3-inhibited zygotes at both establishment and maintenance phase (posterior localization indicated by white arrowheads). In pkc-3 ( RNAi ), PAR-6 is absent from the membrane at all times. PAR-3 (E and F) still polarizes in PKC-3-inhibited zygotes, but becomes weaker and less asymmetric during maintenance phase. Note that (B), (D), and (F) show the same wild-type and TS zygotes with the PAR-3 boundary position in TS indicated (red arrowheads) to allow comparison: PAR-6 and PKC-3 are clearly visible at the posterior membrane (white arrowheads), while PAR-3 is undetectable, as in wild-type. Bright foci in (D) are non-specific centrosome staining. (G and H) Normalized ASI measurements for late establishment phase datasets represented in (A) to (F). ASI is normalized to control (wild-type [WT] or DMSO) for each protein. (I and J) Anterior to posterior membrane distributions of PAR-3 (red) and PKC-3 (black) in wild-type (I) and pkc-3 ( ts ) (J) embryos. Arrows highlight the posterior extension of PKC-3 relative to PAR-3. Mean ± SD is shown. (K) Close-up view of the boundary region showing PAR-3 (top) and PKC-3 (bottom) for one representative zygote for wild-type (WT) and pkc-3 ( ts ) backgrounds as indicated. Dashed rectangular selection denotes regions where PKC-3 is present in absence of PAR-3. (L) PKC-3 to PAR-3 ASI ratio for wild-type (WT) and pkc-3 ( ts ). (M) Dual labeling of PAR-2 and PKC-3 in live, CRT90-treated zygotes (top) and fixed, pkc-3 ( ts ) embryos (bottom) reveal overlap of aPAR and pPAR proteins. ∗∗ p
    Figure Legend Snippet: Membrane Localization of PAR-6/PKC-3 Is Decoupled from PAR-3 when PKC-3 Is Inactive (A–F) Representative midsection confocal images of live and fixed zygotes at establishment and maintenance phase comparing control (DMSO, wild-type), pkc-3 ( RNAi ), and PKC-3-inhibited (CRT90, pkc-3 ( ts )) conditions. PAR-6 (A and B) and PKC-3 (C and D) show loss of asymmetric membrane staining in PKC-3-inhibited zygotes at both establishment and maintenance phase (posterior localization indicated by white arrowheads). In pkc-3 ( RNAi ), PAR-6 is absent from the membrane at all times. PAR-3 (E and F) still polarizes in PKC-3-inhibited zygotes, but becomes weaker and less asymmetric during maintenance phase. Note that (B), (D), and (F) show the same wild-type and TS zygotes with the PAR-3 boundary position in TS indicated (red arrowheads) to allow comparison: PAR-6 and PKC-3 are clearly visible at the posterior membrane (white arrowheads), while PAR-3 is undetectable, as in wild-type. Bright foci in (D) are non-specific centrosome staining. (G and H) Normalized ASI measurements for late establishment phase datasets represented in (A) to (F). ASI is normalized to control (wild-type [WT] or DMSO) for each protein. (I and J) Anterior to posterior membrane distributions of PAR-3 (red) and PKC-3 (black) in wild-type (I) and pkc-3 ( ts ) (J) embryos. Arrows highlight the posterior extension of PKC-3 relative to PAR-3. Mean ± SD is shown. (K) Close-up view of the boundary region showing PAR-3 (top) and PKC-3 (bottom) for one representative zygote for wild-type (WT) and pkc-3 ( ts ) backgrounds as indicated. Dashed rectangular selection denotes regions where PKC-3 is present in absence of PAR-3. (L) PKC-3 to PAR-3 ASI ratio for wild-type (WT) and pkc-3 ( ts ). (M) Dual labeling of PAR-2 and PKC-3 in live, CRT90-treated zygotes (top) and fixed, pkc-3 ( ts ) embryos (bottom) reveal overlap of aPAR and pPAR proteins. ∗∗ p

    Techniques Used: Staining, Selection, Labeling

    Polarization through Coupling of PAR-3- and CDC-42-Dependent aPAR Assemblies (i) PKC-3 kinase activity ensures that PKC-3 loads via a PAR-3 intermediate, in which PKC-3 activity is suppressed. This dependence on PAR-3 can be bypassed upon inhibition of PKC-3 (dashed arrow). (ii) Clustering of membrane-associated PAR-3 allows it to be segregated by cortical flow into the anterior, carrying along associated PKC-3 molecules and generating asymmetric sites for further PKC-3 loading. (iii) PKC-3 activation requires conversion into a CDC-42-associated assembly, which relieves inhibition of PKC-3 by PAR-3. (iv) The CDC-42-dependent module is freely diffusible on the membrane and locally excludes pPARs. (v) Dissociation of CDC-42-dependent assemblies limits the spread of active PKC-3 at the membrane from the PAR-3 recruiting site. (vi) PKC-3 returns to the cytoplasm where it must load again via PAR-3.
    Figure Legend Snippet: Polarization through Coupling of PAR-3- and CDC-42-Dependent aPAR Assemblies (i) PKC-3 kinase activity ensures that PKC-3 loads via a PAR-3 intermediate, in which PKC-3 activity is suppressed. This dependence on PAR-3 can be bypassed upon inhibition of PKC-3 (dashed arrow). (ii) Clustering of membrane-associated PAR-3 allows it to be segregated by cortical flow into the anterior, carrying along associated PKC-3 molecules and generating asymmetric sites for further PKC-3 loading. (iii) PKC-3 activation requires conversion into a CDC-42-associated assembly, which relieves inhibition of PKC-3 by PAR-3. (iv) The CDC-42-dependent module is freely diffusible on the membrane and locally excludes pPARs. (v) Dissociation of CDC-42-dependent assemblies limits the spread of active PKC-3 at the membrane from the PAR-3 recruiting site. (vi) PKC-3 returns to the cytoplasm where it must load again via PAR-3.

    Techniques Used: Activity Assay, Inhibition, Flow Cytometry, Activation Assay

    PKC-3 Inhibition Promotes PAR-3-Independent Formation of CDC-42-Dependent PAR-6/PKC-3 Assemblies (A–C) Representative midsection confocal images of live embryos at maintenance phase showing GFP::PKC-3 (A) or PAR-6::GFP (B and C) of DMSO, CRT90-treated, and pkc-3 ( ts ) zygotes subject to RNAi as indicated. (D) Quantification of rescue for datasets represented in (A) to (C), normalized to membrane signal in control RNAi and CRT90-treated/ pkc-3 ( ts ) zygotes for each dataset. (E) Representative midsection confocal images of wild-type and pkc-3 ( ts ) zygotes during polarity establishment subject to RNAi as indicated and immunostained for PKC-3. (F) Quantification of rescue as measured by anterior domain cortical intensity of PKC-3 for datasets represented in (E). For each zygote, anterior PKC-3 cortical intensity is divided by cytoplasmic intensity. Values greater than 1 indicate presence at the membrane. Mean ± 95% confidence interval (CI) (N) is shown. See STAR Methods for further details. (G) Representative midsection confocal images during polarity establishment of wild-type and pkc-3 ( ts ) embryos upon cgef-1 ( RNAi ), stained for PKC-3. Scatterplot representing the anterior domain cortical intensity of PKC-3 as in (F) in cgef-1 ( RNAi ) and pkc-3 ( ts ); cgef-1 ( RNAi ). Mean ± 95% CI (N) is shown. ∗∗ p
    Figure Legend Snippet: PKC-3 Inhibition Promotes PAR-3-Independent Formation of CDC-42-Dependent PAR-6/PKC-3 Assemblies (A–C) Representative midsection confocal images of live embryos at maintenance phase showing GFP::PKC-3 (A) or PAR-6::GFP (B and C) of DMSO, CRT90-treated, and pkc-3 ( ts ) zygotes subject to RNAi as indicated. (D) Quantification of rescue for datasets represented in (A) to (C), normalized to membrane signal in control RNAi and CRT90-treated/ pkc-3 ( ts ) zygotes for each dataset. (E) Representative midsection confocal images of wild-type and pkc-3 ( ts ) zygotes during polarity establishment subject to RNAi as indicated and immunostained for PKC-3. (F) Quantification of rescue as measured by anterior domain cortical intensity of PKC-3 for datasets represented in (E). For each zygote, anterior PKC-3 cortical intensity is divided by cytoplasmic intensity. Values greater than 1 indicate presence at the membrane. Mean ± 95% confidence interval (CI) (N) is shown. See STAR Methods for further details. (G) Representative midsection confocal images during polarity establishment of wild-type and pkc-3 ( ts ) embryos upon cgef-1 ( RNAi ), stained for PKC-3. Scatterplot representing the anterior domain cortical intensity of PKC-3 as in (F) in cgef-1 ( RNAi ) and pkc-3 ( ts ); cgef-1 ( RNAi ). Mean ± 95% CI (N) is shown. ∗∗ p

    Techniques Used: Inhibition, Staining

    PKC-3 Kinase Inhibition Leads to Symmetric Division and Loss of Asymmetry of Downstream Polarity Markers (A–C) Model for symmetry breaking by the PAR system in C. elegans . aPARs (red) initially occupy the membrane and pPARs (blue) are cytoplasmic (A, Meiosis II). A cue (purple) from the centrosome pair (black spheres) segregates aPARs into the anterior and promotes formation of a posterior PAR domain at the opposite pole (A, Establishment). PAR domains are then stable until cytokinesis (A, Maintenance) and drive polarization of cytoplasmic factors such as MEX-5/6 (green) and P granules (orange), which ensure the daughter cells acquire distinct fates (A, Two-cell). (B) Symmetry breaking can occur in two ways: (i) segregation of aPARs by cortical actomyosin flow (advection); and (ii) posterior PAR-2 loading. (C) A complex network of physical and regulatory interactions links the PAR proteins. Membrane binding (gray lines), physical interactions (black lines), as well as positive (→) and negative (⊥) feedback, are shown. Where links are indirect or unknown, dashed lines are used. Both CDC-42 and PAR-3 are required for stable membrane association of PAR-6/PKC-3. PAR-6 and PKC-3 depend on each other for membrane association. PAR-2, LGL-1, and presumably CHIN-1, are able to load onto the membrane independently. PAR-1 also binds membrane but requires PAR-2 to reach maximal concentrations. PKC-3 phosphorylates PAR-1, PAR-2, and LGL-1 and displaces them from the membrane. Exclusion of CHIN-1 from the anterior is dependent on PKC-3, but whether it is a direct target of PKC-3 is unknown. Together, PAR-1, via phosphorylation of PAR-3, and CHIN-1, by suppressing activated CDC-42, prevent invasion of the posterior domain by aPARs. PAR-3 and PAR-2 have been proposed to undergo oligomerization, which is thought to enhance their membrane association (noted by circular arrows). See recent reviews ( Goehring, 2014 , Hoege and Hyman, 2013 , Motegi and Seydoux, 2013 ) for more information. (D) Midsection confocal images of fixed zygotes stained for PAR-2 at polarity maintenance and two-cell stage comparing wild-type, pkc-3 ( ts ), and pkc-3 ( RNAi ) conditions. (E) Midsection fluorescent images of mCherry:PAR-2-expressing zygotes at maintenance and two-cell stage in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). (F) Midsection (PAR-1, LGL-1, PIE-1, MEX-5) or cortical (CHIN-1) fluorescent images of maintenance-phase zygotes expressing markers to various downstream polarity markers in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). Asymmetry in (D) to (F) is quantified by the asymmetry index, with one being normal asymmetry and zero, no asymmetry (ASI, normalized to DMSO/WT controls). ∗ p
    Figure Legend Snippet: PKC-3 Kinase Inhibition Leads to Symmetric Division and Loss of Asymmetry of Downstream Polarity Markers (A–C) Model for symmetry breaking by the PAR system in C. elegans . aPARs (red) initially occupy the membrane and pPARs (blue) are cytoplasmic (A, Meiosis II). A cue (purple) from the centrosome pair (black spheres) segregates aPARs into the anterior and promotes formation of a posterior PAR domain at the opposite pole (A, Establishment). PAR domains are then stable until cytokinesis (A, Maintenance) and drive polarization of cytoplasmic factors such as MEX-5/6 (green) and P granules (orange), which ensure the daughter cells acquire distinct fates (A, Two-cell). (B) Symmetry breaking can occur in two ways: (i) segregation of aPARs by cortical actomyosin flow (advection); and (ii) posterior PAR-2 loading. (C) A complex network of physical and regulatory interactions links the PAR proteins. Membrane binding (gray lines), physical interactions (black lines), as well as positive (→) and negative (⊥) feedback, are shown. Where links are indirect or unknown, dashed lines are used. Both CDC-42 and PAR-3 are required for stable membrane association of PAR-6/PKC-3. PAR-6 and PKC-3 depend on each other for membrane association. PAR-2, LGL-1, and presumably CHIN-1, are able to load onto the membrane independently. PAR-1 also binds membrane but requires PAR-2 to reach maximal concentrations. PKC-3 phosphorylates PAR-1, PAR-2, and LGL-1 and displaces them from the membrane. Exclusion of CHIN-1 from the anterior is dependent on PKC-3, but whether it is a direct target of PKC-3 is unknown. Together, PAR-1, via phosphorylation of PAR-3, and CHIN-1, by suppressing activated CDC-42, prevent invasion of the posterior domain by aPARs. PAR-3 and PAR-2 have been proposed to undergo oligomerization, which is thought to enhance their membrane association (noted by circular arrows). See recent reviews ( Goehring, 2014 , Hoege and Hyman, 2013 , Motegi and Seydoux, 2013 ) for more information. (D) Midsection confocal images of fixed zygotes stained for PAR-2 at polarity maintenance and two-cell stage comparing wild-type, pkc-3 ( ts ), and pkc-3 ( RNAi ) conditions. (E) Midsection fluorescent images of mCherry:PAR-2-expressing zygotes at maintenance and two-cell stage in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). (F) Midsection (PAR-1, LGL-1, PIE-1, MEX-5) or cortical (CHIN-1) fluorescent images of maintenance-phase zygotes expressing markers to various downstream polarity markers in DMSO (control), CRT90-treated, and pkc-3 ( RNAi ). Asymmetry in (D) to (F) is quantified by the asymmetry index, with one being normal asymmetry and zero, no asymmetry (ASI, normalized to DMSO/WT controls). ∗ p

    Techniques Used: Inhibition, Flow Cytometry, Binding Assay, Staining, Expressing

    PAR-3 and CDC-42 Have Opposing Regulatory Roles in an In Vivo PKC-3 Activity Assay (A) C1B targeting strategy for inducing PKC-3 membrane loading by PMA. PKC-3 kinase activity is monitored by following loss of PAR-2 from the membrane. (B) Zygotes expressing GFP::C1B alone (GFP::C1B-Ø) or GFP::C1B-PKC-3 along with mCherry::PAR-2 were subject to the indicated treatment. Note that uniform membrane targeting of C1B-PKC-3 leads to reduction of PAR-2 domain size, whereas omitting PMA or expressing C1B alone has no effect. Right: cartoon representation of results. (C) Quantification of PAR-2 domain size ratio for embryos shown in (B). (D) PAR-2 retention in GFP::C1B-PKC-3 expressing zygotes treated with PMA and CRT90 confirms that induced PAR-2 loss is dependent on PKC-3 kinase activity. (E) Zygotes expressing mCherry::PAR-2 with GFP::C1B-Ø or GFP::C1B-PKC-3 subject to par-6 , cdc-42 , or par-3 ( RNAi ) before and 5 min after PMA addition. (F) Quantification of PAR-2 cortex retention comparing GFP::C1B-PKC-3 and GFP::C1B-Ø zygotes after treatment with PMA as in (E). Representative midsection confocal images are shown in (B), (D), and (E) before and 5 min after PMA/DMSO addition. ∗∗∗ p
    Figure Legend Snippet: PAR-3 and CDC-42 Have Opposing Regulatory Roles in an In Vivo PKC-3 Activity Assay (A) C1B targeting strategy for inducing PKC-3 membrane loading by PMA. PKC-3 kinase activity is monitored by following loss of PAR-2 from the membrane. (B) Zygotes expressing GFP::C1B alone (GFP::C1B-Ø) or GFP::C1B-PKC-3 along with mCherry::PAR-2 were subject to the indicated treatment. Note that uniform membrane targeting of C1B-PKC-3 leads to reduction of PAR-2 domain size, whereas omitting PMA or expressing C1B alone has no effect. Right: cartoon representation of results. (C) Quantification of PAR-2 domain size ratio for embryos shown in (B). (D) PAR-2 retention in GFP::C1B-PKC-3 expressing zygotes treated with PMA and CRT90 confirms that induced PAR-2 loss is dependent on PKC-3 kinase activity. (E) Zygotes expressing mCherry::PAR-2 with GFP::C1B-Ø or GFP::C1B-PKC-3 subject to par-6 , cdc-42 , or par-3 ( RNAi ) before and 5 min after PMA addition. (F) Quantification of PAR-2 cortex retention comparing GFP::C1B-PKC-3 and GFP::C1B-Ø zygotes after treatment with PMA as in (E). Representative midsection confocal images are shown in (B), (D), and (E) before and 5 min after PMA/DMSO addition. ∗∗∗ p

    Techniques Used: In Vivo, Activity Assay, Expressing

    9) Product Images from "Evidence for Serine/Threonine and Histidine Kinase Activity in the Tobacco Ethylene Receptor Protein NTHK2 1"

    Article Title: Evidence for Serine/Threonine and Histidine Kinase Activity in the Tobacco Ethylene Receptor Protein NTHK2 1

    Journal: Plant Physiology

    doi: 10.1104/pp.103.034686

    Phosphorylation assay of different versions of NTHK2-KD. The phosphorylation was performed in the presence of Ca 2+ (A) or Mn 2+ (B). The NTHK2-KD or other versions (1 μ g) was incubated under phosphorylating conditions with no substrate or with MBP (2 μ g), NTHK2-RD (2 μ g), or GST (2 μ g). NTHK2-RD, MBP, or GST plus MBP were also incubated in the absence of NTHK2-KD and used as controls. The phosphorylated proteins were resolved on SDS-PAGE gel, transferred to PVDF membranes, and subjected to autoradiography (top) or Coomassie blue staining (bottom). C, Effect of N-box and G2-box mutation on the phosphorylation of NTHK2-KD. NTHK2-KD (mN) with N-box mutation and NTHK2-KD (mG2) with G2-box mutation were assayed for their kinase activities following standard method in the presence of Mn 2+ or Ca 2+ . Quantification of the signal intensity was performed using Alpha Innotech Imaging System (Alpha Innotech, San Leandro, CA).
    Figure Legend Snippet: Phosphorylation assay of different versions of NTHK2-KD. The phosphorylation was performed in the presence of Ca 2+ (A) or Mn 2+ (B). The NTHK2-KD or other versions (1 μ g) was incubated under phosphorylating conditions with no substrate or with MBP (2 μ g), NTHK2-RD (2 μ g), or GST (2 μ g). NTHK2-RD, MBP, or GST plus MBP were also incubated in the absence of NTHK2-KD and used as controls. The phosphorylated proteins were resolved on SDS-PAGE gel, transferred to PVDF membranes, and subjected to autoradiography (top) or Coomassie blue staining (bottom). C, Effect of N-box and G2-box mutation on the phosphorylation of NTHK2-KD. NTHK2-KD (mN) with N-box mutation and NTHK2-KD (mG2) with G2-box mutation were assayed for their kinase activities following standard method in the presence of Mn 2+ or Ca 2+ . Quantification of the signal intensity was performed using Alpha Innotech Imaging System (Alpha Innotech, San Leandro, CA).

    Techniques Used: Phosphorylation Assay, Incubation, SDS Page, Autoradiography, Staining, Mutagenesis, Imaging

    10) Product Images from "Na+/K+-ATPase Is Present in Scrapie-Associated Fibrils, Modulates PrP Misfolding In Vitro and Links PrP Function and Dysfunction"

    Article Title: Na+/K+-ATPase Is Present in Scrapie-Associated Fibrils, Modulates PrP Misfolding In Vitro and Links PrP Function and Dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026813

    Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.
    Figure Legend Snippet: Western blotting confirms the presence of ApoE and Na + /K + -ATPase α-chains in SAF preparations. Based on an estimate of 10 µg PrP Sc per brain, the equivalent of 0.25 µg/µl PrP Sc from an ME7 (lane 1), 22F (lane 2) and 79A (lane 3) SAF preparation were resolved by SDS-PAGE, blotted onto PVDF membrane and probed with the primary antibody against (A) Apolipoprotein E (B) Total Na + /K + ATPase α-chains using a pan α-chain antibody (C) Na + /K + ATPase α2 isoform and (D) Na + /K + ATPase α3 isoform. In all cases, in lanes 4 and 5 were loaded the equivalent of 0.25 µg/µl of control preparation material from uninfected WT and PrP −/− mouse brains respectively. Molecular weight markers are in kDa.

    Techniques Used: Western Blot, SDS Page, Molecular Weight

    11) Product Images from "Acute Changes in NADPH Oxidase 4 in Early Post-Traumatic Osteoarthritis"

    Article Title: Acute Changes in NADPH Oxidase 4 in Early Post-Traumatic Osteoarthritis

    Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

    doi: 10.1002/jor.24417

    Kinetics of Nox4 mRNA expression as measured by RT-PCR (A) and protein as measured by western blot (B) after non-invasive ACL rupture of right knees of 10-week old female C57Bl/6 mice compared to control left knees from the same animals. * indicates p
    Figure Legend Snippet: Kinetics of Nox4 mRNA expression as measured by RT-PCR (A) and protein as measured by western blot (B) after non-invasive ACL rupture of right knees of 10-week old female C57Bl/6 mice compared to control left knees from the same animals. * indicates p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay

    μCT assessment of bone density (bone volume/total volume) 7 days after non-invasive joint injury in untreated wild-type mice, Nox4 knockout mice, and wild-type mice treated with the Nox4 inhibitor GKT137831. Mouse knee μCT 3-D reconstructions (top) and graphs of BV/TV of left/right pairs of knees (bottom) from female B57Bl/6 mice 7 days after non-invasive knee injury. Mice were treated with either GKT137831 compound or vehicle by gavage on the day of injury and every day thereafter. n=7 per condition. * indicates p
    Figure Legend Snippet: μCT assessment of bone density (bone volume/total volume) 7 days after non-invasive joint injury in untreated wild-type mice, Nox4 knockout mice, and wild-type mice treated with the Nox4 inhibitor GKT137831. Mouse knee μCT 3-D reconstructions (top) and graphs of BV/TV of left/right pairs of knees (bottom) from female B57Bl/6 mice 7 days after non-invasive knee injury. Mice were treated with either GKT137831 compound or vehicle by gavage on the day of injury and every day thereafter. n=7 per condition. * indicates p

    Techniques Used: Mouse Assay, Knock-Out

    Mechanical testing of young (10 week old) wild type vs. Nox4 knockout mice. Femurs from WT mice are significantly less stiff, with similar ultimate force, modulus, and ultimate stress in 3-point bending tests. * indicates p
    Figure Legend Snippet: Mechanical testing of young (10 week old) wild type vs. Nox4 knockout mice. Femurs from WT mice are significantly less stiff, with similar ultimate force, modulus, and ultimate stress in 3-point bending tests. * indicates p

    Techniques Used: Knock-Out, Mouse Assay

    μCT analysis of young (10 week old) wild type vs. Nox4 knockout mice. Quantification of epiphyseal and metaphysis bone volume over total volume (BV/TV) and trabecular number (Tb. Number), epiphyseal trabecular thickness (Tb. Thickness), and mid-diaphyseal tissue mineral density (TMD) shows significant differences in the knockout mice for all characteristics. * indicates p
    Figure Legend Snippet: μCT analysis of young (10 week old) wild type vs. Nox4 knockout mice. Quantification of epiphyseal and metaphysis bone volume over total volume (BV/TV) and trabecular number (Tb. Number), epiphyseal trabecular thickness (Tb. Thickness), and mid-diaphyseal tissue mineral density (TMD) shows significant differences in the knockout mice for all characteristics. * indicates p

    Techniques Used: Knock-Out, Mouse Assay

    Nox4 activity, protein, and mRNA expression in primary human chondrocytes after cytokine treatment with or without Nox4 inhibition. Kinetics of hydrogen peroxide production (top), Nox4 protein (middle), and Nox4 mRNA expression (bottom) in response to catabolic cytokines: (A) 10 ng/ml IL-1β treatment or (B) 10 ng/ml TNF-α treatment. (C) Kinetics of Nox4 activity and expression after addition of 10 ng/ml TGF-β, an anabolic cytokine. (D) Suppression of the TGF-B mediated increase in hydrogen peroxide production and Nox4 protein levels in response to TGF-β and TGF-β + 1 mM GKT137831 treatment. In all experiments, percent change from baseline H2O2 production was measured by Amplex Red Assay, fold change in mRNA expression was measured by quantitative reverse transcription–polymerase chain reaction, and protein was measured by western blot and is in comparison to vehicle treated cells. * indicates p
    Figure Legend Snippet: Nox4 activity, protein, and mRNA expression in primary human chondrocytes after cytokine treatment with or without Nox4 inhibition. Kinetics of hydrogen peroxide production (top), Nox4 protein (middle), and Nox4 mRNA expression (bottom) in response to catabolic cytokines: (A) 10 ng/ml IL-1β treatment or (B) 10 ng/ml TNF-α treatment. (C) Kinetics of Nox4 activity and expression after addition of 10 ng/ml TGF-β, an anabolic cytokine. (D) Suppression of the TGF-B mediated increase in hydrogen peroxide production and Nox4 protein levels in response to TGF-β and TGF-β + 1 mM GKT137831 treatment. In all experiments, percent change from baseline H2O2 production was measured by Amplex Red Assay, fold change in mRNA expression was measured by quantitative reverse transcription–polymerase chain reaction, and protein was measured by western blot and is in comparison to vehicle treated cells. * indicates p

    Techniques Used: Activity Assay, Expressing, Inhibition, Amplex Red Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    12) Product Images from "The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly"

    Article Title: The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14190

    Identification of a CopZ homologue in R. capsulatus . (A) Amino acid alignment of the R. capsulatus CopZ (R.c.) with the respective CopZ-homologues of Enterococcus hirae (E.h.) Bacillus subtilis (B.s.), Rhodobacter sphaeroides (R.s.), and Homo sapiens (H.s.). The conserved Cu-binding motif is shown in red. (B) Genetic organization of copZ in R. capsulatus . lepA and etp presumably encode for a translation factor and a phosphotyrosine protein phosphatase, respectively. The open reading frames rcc03124 and rcc03126 encode for hypothetical proteins. (C) R. capsulatus cells were grown in MPYE medium, precipitated with trichloroacetic acid and the pellet was dissolved loading buffer. After SDS-PAGE, the gel was either stained directly with coomassie brilliant blue (CBB) as loading control, or was blotted and decorated with α-CopZ antibodies (WB). WT corresponds to MT1131, Δ copZ to a MT1131 derivative carrying an insertion-deletion mutation within copZ and Δ copZ -pcopZ to the Δ copZ strain with a plasmid-encoded copZ . (D) MT1131 (WT) and Δ copZ cell extracts were separated into a soluble fraction and a membrane fraction by ultracentrifugation. Subsequently, the material was separated by SDS-PAGE and decorated with α-CopZ antibodies. (E) The cellular concentration of CopZ in MT1131 grown on MPYE medium without further Cu supplementation was determined by quantitative western blotting, using defined amounts of purified CopZ as reference. Signal intensity was quantified by ImageJ and several independent experiments were performed and a representative western blot is shown. Note that the purified CopZ contained a His-tag and it therefore migrates slower on SDS-PAGE than the native CopZ. (F) RT-PCR analyses of mRNA levels in wild type cells grown on MPYE without and with Cu supplementation (10 μM Cu(II)). A representative gel of three independent experiments is shown. The 16S ribosomal RNA served as control and the ccoI and copA mRNA as reference. Quantification was performed with ImageJ and signal intensity of the mRNA level in cells without Cu supplementation was set to 100%. (G) The CopZ levels in whole cells grown either on enriched medium (MPYE) or minimal medium (MedA) were analysed by immunoblotting as described above. When indicated, CuSO 4 was added to the growth medium. The levels of the Rieske Fe-S protein PetA served as loading control.
    Figure Legend Snippet: Identification of a CopZ homologue in R. capsulatus . (A) Amino acid alignment of the R. capsulatus CopZ (R.c.) with the respective CopZ-homologues of Enterococcus hirae (E.h.) Bacillus subtilis (B.s.), Rhodobacter sphaeroides (R.s.), and Homo sapiens (H.s.). The conserved Cu-binding motif is shown in red. (B) Genetic organization of copZ in R. capsulatus . lepA and etp presumably encode for a translation factor and a phosphotyrosine protein phosphatase, respectively. The open reading frames rcc03124 and rcc03126 encode for hypothetical proteins. (C) R. capsulatus cells were grown in MPYE medium, precipitated with trichloroacetic acid and the pellet was dissolved loading buffer. After SDS-PAGE, the gel was either stained directly with coomassie brilliant blue (CBB) as loading control, or was blotted and decorated with α-CopZ antibodies (WB). WT corresponds to MT1131, Δ copZ to a MT1131 derivative carrying an insertion-deletion mutation within copZ and Δ copZ -pcopZ to the Δ copZ strain with a plasmid-encoded copZ . (D) MT1131 (WT) and Δ copZ cell extracts were separated into a soluble fraction and a membrane fraction by ultracentrifugation. Subsequently, the material was separated by SDS-PAGE and decorated with α-CopZ antibodies. (E) The cellular concentration of CopZ in MT1131 grown on MPYE medium without further Cu supplementation was determined by quantitative western blotting, using defined amounts of purified CopZ as reference. Signal intensity was quantified by ImageJ and several independent experiments were performed and a representative western blot is shown. Note that the purified CopZ contained a His-tag and it therefore migrates slower on SDS-PAGE than the native CopZ. (F) RT-PCR analyses of mRNA levels in wild type cells grown on MPYE without and with Cu supplementation (10 μM Cu(II)). A representative gel of three independent experiments is shown. The 16S ribosomal RNA served as control and the ccoI and copA mRNA as reference. Quantification was performed with ImageJ and signal intensity of the mRNA level in cells without Cu supplementation was set to 100%. (G) The CopZ levels in whole cells grown either on enriched medium (MPYE) or minimal medium (MedA) were analysed by immunoblotting as described above. When indicated, CuSO 4 was added to the growth medium. The levels of the Rieske Fe-S protein PetA served as loading control.

    Techniques Used: Binding Assay, SDS Page, Staining, Western Blot, Mutagenesis, Plasmid Preparation, Concentration Assay, Purification, Reverse Transcription Polymerase Chain Reaction

    CopZ is a Cu binding protein that forms redox-sensitive oligomers. (A) Purified CopZ was separated on native, non-reducing PAGE and after western transfer was decorated with α-CopZ antibodies (left panel). The dominant CopZ species are indicated by numbers (1–3); two additional minor species are indicated by (*), but those were not further analyzed. The dominant species 1–3 were gel-extracted from the native gel and separated on a second dimension SDS-PAGE under reducing conditions (right panel). The second dimension was stained with coomassie brilliant blue. Indicated are CopZ and a second band (?) that was not characterized further. (B) Purified CopZ and its derivatives lacking either one (C10S) or both conserved cysteines of the Cu binding motif (C10S/C13S) were separated on native-PAGE either in the absence of the reducing agent DTT or in its presence. Indicated are the different oligomeric states of CopZ. (C) The Cu:protein ratio of purified CopZ was analyzed by atomic absorption spectroscopy. Wild type CopZ or the CopZ(C10S-C13S) mutant that lacked both conserved cysteine residues of the Cu binding motif, were directly analyzed (-Cu) or only after prior incubation with a 5-fold molar excess of Cu(I) and subsequent removal of unbound Cu(I) by gel filtration (+Cu). The molar Cu content was quantified by using a standard curve and the molar Cu:protein ratio was calculated. The values shown represent the mean of three independent experiments and the standard deviation is indicated by error bars.
    Figure Legend Snippet: CopZ is a Cu binding protein that forms redox-sensitive oligomers. (A) Purified CopZ was separated on native, non-reducing PAGE and after western transfer was decorated with α-CopZ antibodies (left panel). The dominant CopZ species are indicated by numbers (1–3); two additional minor species are indicated by (*), but those were not further analyzed. The dominant species 1–3 were gel-extracted from the native gel and separated on a second dimension SDS-PAGE under reducing conditions (right panel). The second dimension was stained with coomassie brilliant blue. Indicated are CopZ and a second band (?) that was not characterized further. (B) Purified CopZ and its derivatives lacking either one (C10S) or both conserved cysteines of the Cu binding motif (C10S/C13S) were separated on native-PAGE either in the absence of the reducing agent DTT or in its presence. Indicated are the different oligomeric states of CopZ. (C) The Cu:protein ratio of purified CopZ was analyzed by atomic absorption spectroscopy. Wild type CopZ or the CopZ(C10S-C13S) mutant that lacked both conserved cysteine residues of the Cu binding motif, were directly analyzed (-Cu) or only after prior incubation with a 5-fold molar excess of Cu(I) and subsequent removal of unbound Cu(I) by gel filtration (+Cu). The molar Cu content was quantified by using a standard curve and the molar Cu:protein ratio was calculated. The values shown represent the mean of three independent experiments and the standard deviation is indicated by error bars.

    Techniques Used: Binding Assay, Purification, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page, Staining, Clear Native PAGE, Atomic Absorption Spectroscopy, Mutagenesis, Incubation, Filtration, Standard Deviation

    CopZ forms a complex with the P 1B -type ATPase CcoI. (A) ICMs of the indicated strains were solubilized, and separated without further purification by BN-PAGE. After western transfer, potential CcoI and CopA complexes were visualized with antibodies against their C-terminal Myc-tags. (B) As in (A), but probing for CcoI complexes in membranes of the Δ copZ strain. (C) ICMs of the Δ ccoI strain complemented with p ccoI (upper two panels), the Δ ccoI strain (middle two panels) and of Δ copZ p ccoI (lower two panels) were solubilized and separated on BN-PAGE as in (A). The BN-PAGE gel lane was cut out, subsequently equilibrated in equilibration buffer containing SDS and urea and subjected to a 2 nd dimension SDS-PAGE. After western transfer, the membrane was horizontally cut and the upper part was decorated with α-Myc antibodies and the lower part with α-CopZ antibodies. Representative gels of at least three biological replicates are shown.
    Figure Legend Snippet: CopZ forms a complex with the P 1B -type ATPase CcoI. (A) ICMs of the indicated strains were solubilized, and separated without further purification by BN-PAGE. After western transfer, potential CcoI and CopA complexes were visualized with antibodies against their C-terminal Myc-tags. (B) As in (A), but probing for CcoI complexes in membranes of the Δ copZ strain. (C) ICMs of the Δ ccoI strain complemented with p ccoI (upper two panels), the Δ ccoI strain (middle two panels) and of Δ copZ p ccoI (lower two panels) were solubilized and separated on BN-PAGE as in (A). The BN-PAGE gel lane was cut out, subsequently equilibrated in equilibration buffer containing SDS and urea and subjected to a 2 nd dimension SDS-PAGE. After western transfer, the membrane was horizontally cut and the upper part was decorated with α-Myc antibodies and the lower part with α-CopZ antibodies. Representative gels of at least three biological replicates are shown.

    Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page

    13) Product Images from "Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35"

    Article Title: Amyloid properties of the yeast cell wall protein Toh1 and its interaction with prion proteins Rnq1 and Sup35

    Journal: Prion

    doi: 10.1080/19336896.2018.1558763

    Toh1-YFP protein forms detergent-resistant aggregates in yeast cells that are revealed predominantly in the cell debris. (a) Localization of the Toh1-YFP aggregates in yeast cells. Transformants of BY4742 strain expressing TOH1-YFP and GFP genes were grown on -Leu selective media for 48 h prior to fluorescence microscopy. In the case of the constructs under P CUP1 promoter media was supplemented with 100 μM CuSO 4 . (b) Fractionation of the crude cell lysates extracted from BY4742 cells expressing the Toh1-YFP protein showing predominating localization of Toh1-YFP in the debris fraction. Cells expressing Toh1-YFP protein were harvested after 48h, lysed and the crude cell lysate was separated into debris and lysate fractions followed by the denaturating treatment of the samples (2,5% SDS, 95°C). Samples were resolved in 10% SDS-PAGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). (c) SDD-AGE of the crude protein lysates extracted from BY4742 cells expressing Toh1-YFP protein showing the capacity of Toh1-YFP to form SDS-resistant aggregates. The debris fractions of the crude protein lysates were incubated under semi-denaturing (SD, 1% SDS at room temperature) and denaturing (D, 2,5% SDS, 95°C) conditions. Samples were resolved on 1,5% SDS-AGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). Cells expressing GFP were used as a negative control for aggregation.
    Figure Legend Snippet: Toh1-YFP protein forms detergent-resistant aggregates in yeast cells that are revealed predominantly in the cell debris. (a) Localization of the Toh1-YFP aggregates in yeast cells. Transformants of BY4742 strain expressing TOH1-YFP and GFP genes were grown on -Leu selective media for 48 h prior to fluorescence microscopy. In the case of the constructs under P CUP1 promoter media was supplemented with 100 μM CuSO 4 . (b) Fractionation of the crude cell lysates extracted from BY4742 cells expressing the Toh1-YFP protein showing predominating localization of Toh1-YFP in the debris fraction. Cells expressing Toh1-YFP protein were harvested after 48h, lysed and the crude cell lysate was separated into debris and lysate fractions followed by the denaturating treatment of the samples (2,5% SDS, 95°C). Samples were resolved in 10% SDS-PAGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). (c) SDD-AGE of the crude protein lysates extracted from BY4742 cells expressing Toh1-YFP protein showing the capacity of Toh1-YFP to form SDS-resistant aggregates. The debris fractions of the crude protein lysates were incubated under semi-denaturing (SD, 1% SDS at room temperature) and denaturing (D, 2,5% SDS, 95°C) conditions. Samples were resolved on 1,5% SDS-AGE and immunoblotted using anti-GFP antibody (ab32146 (Abcam, Great Britain)). Cells expressing GFP were used as a negative control for aggregation.

    Techniques Used: Expressing, Fluorescence, Microscopy, Construct, Fractionation, SDS Page, Incubation, Negative Control

    14) Product Images from "Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins"

    Article Title: Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins

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

    doi:

    Rapamycin increases the mobility of the transcriptional regulator Ure2p by inhibiting Tor proteins. Anti-Ure2p immunoblots of whole cell lysates were performed as described in Materials and Methods . ( A ) Jk9–3d a cells were treated with either 100 nM rapamycin or 100 nM FK506 for 15 min. ( B ) CY5754 cells were treated with 20 nM rapamycin for 5 min. ( C ) Cells expressing a mutant form of TOR1 (Ser1972→Thr) that is unable to bind rapamycin and the wild-type parental strain were exposed to 50 nM rapamycin for 30 min.
    Figure Legend Snippet: Rapamycin increases the mobility of the transcriptional regulator Ure2p by inhibiting Tor proteins. Anti-Ure2p immunoblots of whole cell lysates were performed as described in Materials and Methods . ( A ) Jk9–3d a cells were treated with either 100 nM rapamycin or 100 nM FK506 for 15 min. ( B ) CY5754 cells were treated with 20 nM rapamycin for 5 min. ( C ) Cells expressing a mutant form of TOR1 (Ser1972→Thr) that is unable to bind rapamycin and the wild-type parental strain were exposed to 50 nM rapamycin for 30 min.

    Techniques Used: Western Blot, Expressing, Mutagenesis

    15) Product Images from "Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells"

    Article Title: Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

    Journal: Retrovirology

    doi: 10.1186/1742-4690-5-115

    2D gel electrophoresis analysis of recombinant RT identifies protein isoforms . Recombinant RT LAI + GAPDH protein (3 μg each) was solubilised in 2D gel electrophoresis buffer, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel then resolved on a 10% acrylamide SDS-PAGE gel followed by transfer to PVDF membranes. RT was detected by Western blot using an anti-RT antibody (upper panel) and GAPDH detected by Coomassie stain (lower panel). RT isoforms are designated by black arrows and calculated pI indicated. Position of triangles (Δ) denote the reference marks used for calculation of pI.
    Figure Legend Snippet: 2D gel electrophoresis analysis of recombinant RT identifies protein isoforms . Recombinant RT LAI + GAPDH protein (3 μg each) was solubilised in 2D gel electrophoresis buffer, focussed on a pH 7–11 non-linear, 11 cm Immobiline DryStrip gel then resolved on a 10% acrylamide SDS-PAGE gel followed by transfer to PVDF membranes. RT was detected by Western blot using an anti-RT antibody (upper panel) and GAPDH detected by Coomassie stain (lower panel). RT isoforms are designated by black arrows and calculated pI indicated. Position of triangles (Δ) denote the reference marks used for calculation of pI.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Recombinant, SDS Page, Western Blot, Staining

    16) Product Images from "Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC)"

    Article Title: Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC)

    Journal: Scientific Reports

    doi: 10.1038/srep10897

    Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.
    Figure Legend Snippet: Dot blotting analysis for binding site confirmation. Dot blotting was performed to define the geometric orientations of the selected aptamers. ( a ) LCN2 (74.2 pmol) with 9 selected aptamers (LCN2_Apta1 to LCN2_Apta9, 742 pmol) and controls (C1 to C4) were dotted onto the Hybond-P PVDF membrane. After incubation with the HRP-conjugated anti-LCN2 polyclonal antibody, an assay image was taken using an ECL assay protocol. The plotted signal intensities were calculated using ImageJ software and normalized to C2 (LCN2 74.2 pmol). The schematic epitope binding of two aptamers (LCN2_apta2 and LCN2_apta4) is illustrated in ( b ). All parts of this figure were drawn by the authors K-A. L. and J-Y. A.

    Techniques Used: Binding Assay, Incubation, Software

    17) Product Images from "Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex"

    Article Title: Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr705

    Phosphorylation of P20. ( A ) In vitro phosphorylation. Purified rP20 was co-incubated in protein extracts of BaMV and satBaMV co-infected N. benthamiana leaves in the absence or presence of 100 mM EDTA and [γ- 32 P]ATP or [γ- 32 P]GTP and analyzed by 12.5% SDS–PAGE and autoradiography. Reaction mixture lacking rP20 or leaf protein extract served as controls. ( C ) In vivo phosphorylation. N. benthamiana protoplasts were mock-inoculated, inoculated with BaMV viral RNA alone or co-inoculated with BaMV RNA and satBaMV RNA transcripts by electroporation and cultured with [ 33 P]orthophosphate. After 20 h inoculation, protoplasts were harvested, lysed, total proteins were immunoprecipitated with anti-P20 serum, analyzed by 12.5% SDS–PAGE and detected by autoradiography. ( B and D ), Immunodetection of P20 phosphorylated in vitro and in vivo with anti-P20 serum.
    Figure Legend Snippet: Phosphorylation of P20. ( A ) In vitro phosphorylation. Purified rP20 was co-incubated in protein extracts of BaMV and satBaMV co-infected N. benthamiana leaves in the absence or presence of 100 mM EDTA and [γ- 32 P]ATP or [γ- 32 P]GTP and analyzed by 12.5% SDS–PAGE and autoradiography. Reaction mixture lacking rP20 or leaf protein extract served as controls. ( C ) In vivo phosphorylation. N. benthamiana protoplasts were mock-inoculated, inoculated with BaMV viral RNA alone or co-inoculated with BaMV RNA and satBaMV RNA transcripts by electroporation and cultured with [ 33 P]orthophosphate. After 20 h inoculation, protoplasts were harvested, lysed, total proteins were immunoprecipitated with anti-P20 serum, analyzed by 12.5% SDS–PAGE and detected by autoradiography. ( B and D ), Immunodetection of P20 phosphorylated in vitro and in vivo with anti-P20 serum.

    Techniques Used: In Vitro, Purification, Incubation, Infection, SDS Page, Autoradiography, In Vivo, Electroporation, Cell Culture, Immunoprecipitation, Immunodetection

    18) Product Images from "Interleukin33 Is Required for Disposal of Unnecessary Cells during Ovarian Atresia through Regulation of Autophagy and Macrophage Migration"

    Article Title: Interleukin33 Is Required for Disposal of Unnecessary Cells during Ovarian Atresia through Regulation of Autophagy and Macrophage Migration

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1402503

    Diminished autophagy in atretic follicles in Il33 −/− mice. ( A ) Western blot detection of ovarian LC3 in four representative WT or Il33 −/− mice. Notice lower quantities of LC3-II in Il33 −/− ovaries than those
    Figure Legend Snippet: Diminished autophagy in atretic follicles in Il33 −/− mice. ( A ) Western blot detection of ovarian LC3 in four representative WT or Il33 −/− mice. Notice lower quantities of LC3-II in Il33 −/− ovaries than those

    Techniques Used: Mouse Assay, Western Blot

    19) Product Images from "The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase"

    Article Title: The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2009.04.003

    Models for LAF-1 regulation of tra-2
    Figure Legend Snippet: Models for LAF-1 regulation of tra-2

    Techniques Used:

    laf-1 /+ heterozygotes express elevated quantities of TRA-2
    Figure Legend Snippet: laf-1 /+ heterozygotes express elevated quantities of TRA-2

    Techniques Used:

    20) Product Images from "Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein"

    Article Title: Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1338221

    Arsenic inhibits Cdk2 kinase activity and the expression of the phosphatase Cdc25a. (A) Cdk2 kinase was immunoprecipitated from MCF-7 cells after no treatment or a 14 h treatment with 5 nM E2 or 5 nM E2 + 5 µM iAs. The immunoprecipitated protein was incubated with [γ 32 P] ATP and 1 µg histone H1 in a kinase assay. Proteins were then separated by gel electrophoresis, transferred to a filter and visualized by PhosphorImager analysis (phosphor histone H1). The filter was then subjected to western blot analysis with an antibody to Cdk2. (B) Quiescent cells were treated with 5 nM E2 ± 5 µM iAs, mRNA was isolated and qRT-PCR was done with primers to Cdc25a. Representative experiment repeated 4 times as above ( n = 3 ; Error bars = SEM; *p-Value
    Figure Legend Snippet: Arsenic inhibits Cdk2 kinase activity and the expression of the phosphatase Cdc25a. (A) Cdk2 kinase was immunoprecipitated from MCF-7 cells after no treatment or a 14 h treatment with 5 nM E2 or 5 nM E2 + 5 µM iAs. The immunoprecipitated protein was incubated with [γ 32 P] ATP and 1 µg histone H1 in a kinase assay. Proteins were then separated by gel electrophoresis, transferred to a filter and visualized by PhosphorImager analysis (phosphor histone H1). The filter was then subjected to western blot analysis with an antibody to Cdk2. (B) Quiescent cells were treated with 5 nM E2 ± 5 µM iAs, mRNA was isolated and qRT-PCR was done with primers to Cdc25a. Representative experiment repeated 4 times as above ( n = 3 ; Error bars = SEM; *p-Value

    Techniques Used: Activity Assay, Expressing, Immunoprecipitation, Incubation, Kinase Assay, Nucleic Acid Electrophoresis, Western Blot, Isolation, Quantitative RT-PCR

    21) Product Images from "Differential Regulation of a Novel Variant of the α6 Integrin, α6p"

    Article Title: Differential Regulation of a Novel Variant of the α6 Integrin, α6p

    Journal: Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research

    doi:

    Disruption of the actin cytoskeleton significantly reduced cell surface expression of α 6 , α 6p , and β 1 integrins. Surface changes of α 6 , β 1 , and α 6p were determined by surface of DU145H cells with biotin before treatment with either 10 μ M cytochalasin D or 8 μ M nocodazole for 18 h. Labeled cells were lysed, and 200 μ g of total protein were used for immunoprecipitations with anti- α 6 integrin antibody, J1B5. Samples were separated on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by incubation with HRP conjugated to streptavidin ( A ). Resulting α 6 , β 1 , and α 6p integrin protein bands were quantified and graphed ( B ).
    Figure Legend Snippet: Disruption of the actin cytoskeleton significantly reduced cell surface expression of α 6 , α 6p , and β 1 integrins. Surface changes of α 6 , β 1 , and α 6p were determined by surface of DU145H cells with biotin before treatment with either 10 μ M cytochalasin D or 8 μ M nocodazole for 18 h. Labeled cells were lysed, and 200 μ g of total protein were used for immunoprecipitations with anti- α 6 integrin antibody, J1B5. Samples were separated on a 7.5% polyacrylamide gel under nonreducing conditions. Proteins were transferred to PVDF membrane followed by incubation with HRP conjugated to streptavidin ( A ). Resulting α 6 , β 1 , and α 6p integrin protein bands were quantified and graphed ( B ).

    Techniques Used: Expressing, Labeling, Incubation

    22) Product Images from "Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae"

    Article Title: Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006504

    [ NSI + ] strain contains [ PIN + ] prion which acts as the enhancer of the nonsense suppression. (A) SDD-AGE of protein lysates extracted from the 1-1-D931 [ NSI + ] and 1-1-1-D931 [ nsi - ] strains expressing pCUP1-RNQ1-CFP(LEU2) plasmid. Protein lysates were treated with 1% SDS at room temperature. SDS-resistant aggregates of Rnq1-CFP were detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) The effects of RNQ1 deletion on the [ NSI + ] phenotypic manifestation. RNQ1 deletion was obtained as described in Materials and Methods. The [ nsi - ] and [ nsi - ] rnq Δ strains were obtained from the corresponding [ NSI + ] strains by GuHCl treatment. To express RNQ1 , the 5-1-1-D931 [ NSI + ] rnq Δ strain and its [ nsi - ] derivative were transformed with the YGPM25a02 plasmid containing a genomic fragment encoding RNQ1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade medium or–Leu medium with 150 μM CuSO 4 containing galactose as the sole carbon source. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.
    Figure Legend Snippet: [ NSI + ] strain contains [ PIN + ] prion which acts as the enhancer of the nonsense suppression. (A) SDD-AGE of protein lysates extracted from the 1-1-D931 [ NSI + ] and 1-1-1-D931 [ nsi - ] strains expressing pCUP1-RNQ1-CFP(LEU2) plasmid. Protein lysates were treated with 1% SDS at room temperature. SDS-resistant aggregates of Rnq1-CFP were detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) The effects of RNQ1 deletion on the [ NSI + ] phenotypic manifestation. RNQ1 deletion was obtained as described in Materials and Methods. The [ nsi - ] and [ nsi - ] rnq Δ strains were obtained from the corresponding [ NSI + ] strains by GuHCl treatment. To express RNQ1 , the 5-1-1-D931 [ NSI + ] rnq Δ strain and its [ nsi - ] derivative were transformed with the YGPM25a02 plasmid containing a genomic fragment encoding RNQ1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade medium or–Leu medium with 150 μM CuSO 4 containing galactose as the sole carbon source. Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Transformation Assay, Incubation

    Mit1 is not a determinant of the [ NSI + ] factor. (A) SDD-AGE assay of protein lysates extracted from the 4-1-1-D931 [ NSI + ] and 1-4-1-1-D931 [ nsi - ] strains expressing pMIT1-MIT1-GFP(URA3) plasmid. Cells were grown for 48 h at 30°C in liquid–Ura selective medium containing 150 μM CuSO 4 . Protein lysates were treated with 1% SDS at room temperature. Mit1-GFP was detected with monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) MIT1 deletion does not affect the [ NSI + ] phenotypic manifestation. MIT1 deletion was obtained as described in Materials and Methods. The [ nsi - ] derivative of the 2–936 [ NSI + ] mit1 Δ was obtained by GuHCl treatment. To express MIT1 , 2–936 [ NSI + ] mit1 Δ strain was transformed with YGPM21o12 plasmid from the YSC4613 genomic library containing a genomic fragment encoding MIT1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with a vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO 4 . Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.
    Figure Legend Snippet: Mit1 is not a determinant of the [ NSI + ] factor. (A) SDD-AGE assay of protein lysates extracted from the 4-1-1-D931 [ NSI + ] and 1-4-1-1-D931 [ nsi - ] strains expressing pMIT1-MIT1-GFP(URA3) plasmid. Cells were grown for 48 h at 30°C in liquid–Ura selective medium containing 150 μM CuSO 4 . Protein lysates were treated with 1% SDS at room temperature. Mit1-GFP was detected with monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (B) MIT1 deletion does not affect the [ NSI + ] phenotypic manifestation. MIT1 deletion was obtained as described in Materials and Methods. The [ nsi - ] derivative of the 2–936 [ NSI + ] mit1 Δ was obtained by GuHCl treatment. To express MIT1 , 2–936 [ NSI + ] mit1 Δ strain was transformed with YGPM21o12 plasmid from the YSC4613 genomic library containing a genomic fragment encoding MIT1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with a vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO 4 . Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Transformation Assay, Incubation

    [ SWI + ] prion is a key determinant of nonsense suppression in [ NSI + ] strains. (A) Sedimentation analysis of Swi1(1–297)-YFP protein from the 4-1-1-D931 [ NSI + ] and 1-4-1-1-D931 [ nsi - ] strains expressing pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). Next, SDD-AGE analysis of insoluble fractions of [ NSI + ] and [ nsi - ] strains comprising Swi1(1–297)-YFP was performed. (B) The effects of SWI1 deletion on the [ NSI + ] phenotypic manifestation. SWI1 deletion was obtained as described in Materials and Methods. To express SWI1 , the 11-1-1-D931 [ NSI + ] swi1 Δ strain was transformed with the YGPM19p21 plasmid from the YSC4613 genomic library, containing a genomic fragment encoding SWI1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO 4 . Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.
    Figure Legend Snippet: [ SWI + ] prion is a key determinant of nonsense suppression in [ NSI + ] strains. (A) Sedimentation analysis of Swi1(1–297)-YFP protein from the 4-1-1-D931 [ NSI + ] and 1-4-1-1-D931 [ nsi - ] strains expressing pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). Next, SDD-AGE analysis of insoluble fractions of [ NSI + ] and [ nsi - ] strains comprising Swi1(1–297)-YFP was performed. (B) The effects of SWI1 deletion on the [ NSI + ] phenotypic manifestation. SWI1 deletion was obtained as described in Materials and Methods. To express SWI1 , the 11-1-1-D931 [ NSI + ] swi1 Δ strain was transformed with the YGPM19p21 plasmid from the YSC4613 genomic library, containing a genomic fragment encoding SWI1 under the control of its endogenous promoter. Other strains presented in this Figure were transformed with an empty vector expressing only the LEU2 gene. Transformants were selected on–Leu medium with 150 μM CuSO 4 and replica-plated on–Leu–Ade or–Leu Gal media with 150 μM CuSO 4 . Images were taken after 5 days of incubation of–Ade plates or after 3 passages on Gal plates.

    Techniques Used: Sedimentation, Expressing, Plasmid Preparation, Western Blot, Transformation Assay, Incubation

    [ SWI + ] and [ PIN + ] prions demonstrate complementary interaction. (A) Comparative analysis of the growth of strains containing combinations of [ prion - ] or [ PRION + ] states for Rnq1 and Swi1 as well as deletions of the corresponding genes. “1”–The [ SWI + ][ pin - ] and [ swi - ][ PIN + ] strains were obtained from the 1-1-D931 [ SWI + ][ PIN + ] strain by deletion with subsequent reintroduction of RNQ1 and SWI1 genes, respectively (see “ Materials and Methods ”). The [ swi - ][ pin - ] strain was obtained from the 1-1-D931 [ SWI + ][ PIN + ] strain by GuHCl curing. “2”–The 26-1-4-1-1-D931 [ swi - ][ PIN + ], 12-1-4-1-1-D931 [ SWI + ][ pin - ], and 16-1-4-1-1-D931 [ SWI + ][ PIN + ] strains were obtained by transformation of the 1-4-1-1-D931 [ swi - ][ pin - ] recipient yeast cells with the 1-1-D931 [ SWI + ][ PIN + ] protein lysates followed by analysis of [ SWI ] and [ PIN ] status of the cells as described in “Materials and Methods”. Images were obtained after 5 days of incubation on–Ade plates with 150 μM CuSO 4 or after 3 passages on Gal plates. (B) Sedimentation analysis of Swi1(1–297)-YFP protein from the 16-1-4-1-1-D931 [ SWI + ][ PIN + ] and 12-1-4-1-1-D931 [ SWI + ][ pin - ] strains expressing the pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (C) Analysis of the colocalization of Swi1-YFP and Rnq1-CFP aggregates. The cells of the D938 [ SWI + ][ PIN + ] strain were co-transformed with p426GPD–SWI1YFP and pCUP1-RNQ1-CFP(LEU2) plasmids. Transformants were selected on–Ura–Leu selective media with 150 μM CuSO 4 and incubated for 48 h prior to fluorescence microscopy.
    Figure Legend Snippet: [ SWI + ] and [ PIN + ] prions demonstrate complementary interaction. (A) Comparative analysis of the growth of strains containing combinations of [ prion - ] or [ PRION + ] states for Rnq1 and Swi1 as well as deletions of the corresponding genes. “1”–The [ SWI + ][ pin - ] and [ swi - ][ PIN + ] strains were obtained from the 1-1-D931 [ SWI + ][ PIN + ] strain by deletion with subsequent reintroduction of RNQ1 and SWI1 genes, respectively (see “ Materials and Methods ”). The [ swi - ][ pin - ] strain was obtained from the 1-1-D931 [ SWI + ][ PIN + ] strain by GuHCl curing. “2”–The 26-1-4-1-1-D931 [ swi - ][ PIN + ], 12-1-4-1-1-D931 [ SWI + ][ pin - ], and 16-1-4-1-1-D931 [ SWI + ][ PIN + ] strains were obtained by transformation of the 1-4-1-1-D931 [ swi - ][ pin - ] recipient yeast cells with the 1-1-D931 [ SWI + ][ PIN + ] protein lysates followed by analysis of [ SWI ] and [ PIN ] status of the cells as described in “Materials and Methods”. Images were obtained after 5 days of incubation on–Ade plates with 150 μM CuSO 4 or after 3 passages on Gal plates. (B) Sedimentation analysis of Swi1(1–297)-YFP protein from the 16-1-4-1-1-D931 [ SWI + ][ PIN + ] and 12-1-4-1-1-D931 [ SWI + ][ pin - ] strains expressing the pCUP1-SWI1(1–297)-YFP (URA3) plasmid. Soluble (S) and insoluble (I) fractions were obtained as indicated in Materials and Methods. Swi1(1–297)-YFP was detected using monoclonal rabbit primary antibodies against GFP [E385] (ab32146) (Abcam, Great Britain) and ECL Prime Western Blotting Detection Reagent kit (GE Healthcare, USA). (C) Analysis of the colocalization of Swi1-YFP and Rnq1-CFP aggregates. The cells of the D938 [ SWI + ][ PIN + ] strain were co-transformed with p426GPD–SWI1YFP and pCUP1-RNQ1-CFP(LEU2) plasmids. Transformants were selected on–Ura–Leu selective media with 150 μM CuSO 4 and incubated for 48 h prior to fluorescence microscopy.

    Techniques Used: Transformation Assay, Incubation, Sedimentation, Expressing, Plasmid Preparation, Western Blot, Fluorescence, Microscopy

    23) Product Images from "S-layer Surface-Accessible and Concanavalin A Binding Proteins of Methanosarcina acetivorans and Methanosarcina mazei"

    Article Title: S-layer Surface-Accessible and Concanavalin A Binding Proteins of Methanosarcina acetivorans and Methanosarcina mazei

    Journal: Journal of proteome research

    doi: 10.1021/pr800923e

    Streptavidin-bound M. acetivorans proteins treated with PNGase F, eluted, and resolved by SDS-PAGE. A) SYPRO-Ruby staining reveals total proteins, and B) Near-western blot probed with streptavidin-HRP reveals biotin-tagged proteins. (M) Biotinylated protein markers; ( 1 ) Protein eluate from beads untreated by PNGase F (negative control); ( 2 ) Protein eluate from beads treated with PNGase F. Black Box : MA0829 bands pre-treatment. Upper Black Arrow : MA0829 post-treatment. Lower Black Arrow : PNGase F.
    Figure Legend Snippet: Streptavidin-bound M. acetivorans proteins treated with PNGase F, eluted, and resolved by SDS-PAGE. A) SYPRO-Ruby staining reveals total proteins, and B) Near-western blot probed with streptavidin-HRP reveals biotin-tagged proteins. (M) Biotinylated protein markers; ( 1 ) Protein eluate from beads untreated by PNGase F (negative control); ( 2 ) Protein eluate from beads treated with PNGase F. Black Box : MA0829 bands pre-treatment. Upper Black Arrow : MA0829 post-treatment. Lower Black Arrow : PNGase F.

    Techniques Used: SDS Page, Staining, Western Blot, Negative Control

    24) Product Images from "Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility 1"

    Article Title: Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility 1

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.112.107128

    TEM of sperm from Eno4 Gt/Gt and WT mice. Sperm collected from cauda epididymis were fixed, sectioned, and examined by TEM. A and C ) Sperm from Eno4 Gt/Gt mouse. B ) Sperm from WT mouse. R, ribs of the FS; AN, annulus. Bars = 0.5 μm.
    Figure Legend Snippet: TEM of sperm from Eno4 Gt/Gt and WT mice. Sperm collected from cauda epididymis were fixed, sectioned, and examined by TEM. A and C ) Sperm from Eno4 Gt/Gt mouse. B ) Sperm from WT mouse. R, ribs of the FS; AN, annulus. Bars = 0.5 μm.

    Techniques Used: Transmission Electron Microscopy, Mouse Assay

    Coimmunoprecipitation assay with ENO4, AKAP4, ENO, and PGAM2. A ) Regions of ENO4_V1 (aa 1–571), truncated N-terminal ENO4-N (aa 1–228), and ENO4-C (aa 228–618) used for in vitro synthesized [ 35 S]-labeled probes. B ) [ 35 S]-ENO4_V1
    Figure Legend Snippet: Coimmunoprecipitation assay with ENO4, AKAP4, ENO, and PGAM2. A ) Regions of ENO4_V1 (aa 1–571), truncated N-terminal ENO4-N (aa 1–228), and ENO4-C (aa 228–618) used for in vitro synthesized [ 35 S]-labeled probes. B ) [ 35 S]-ENO4_V1

    Techniques Used: Co-Immunoprecipitation Assay, In Vitro, Synthesized, Labeling

    β-galactosidase activity and ATP levels in Eno4 Gt/Gt and WT testes. A ) β-galactosidase activity was detected in germ cells in testis from Eno4 Gt/Gt mice by X-gal staining. B ) No β-galactosidase activity was detected in testis from
    Figure Legend Snippet: β-galactosidase activity and ATP levels in Eno4 Gt/Gt and WT testes. A ) β-galactosidase activity was detected in germ cells in testis from Eno4 Gt/Gt mice by X-gal staining. B ) No β-galactosidase activity was detected in testis from

    Techniques Used: Activity Assay, Mouse Assay, Staining

    Testis morphology in Eno4 Gt/Gt mice. A ) Hematoxylin and eosin staining in testis of Eno4 Gt/Gt mice. Lobular structures (arrows) were observed along lumen of testis of Eno4 Gt/Gt mice. Bar = 25 μm. B , C , and D ) Testes were fixed, sectioned, and
    Figure Legend Snippet: Testis morphology in Eno4 Gt/Gt mice. A ) Hematoxylin and eosin staining in testis of Eno4 Gt/Gt mice. Lobular structures (arrows) were observed along lumen of testis of Eno4 Gt/Gt mice. Bar = 25 μm. B , C , and D ) Testes were fixed, sectioned, and

    Techniques Used: Mouse Assay, Staining

    SEM of sperm from Eno4 Gt/Gt and WT mice. A and D ) Sperm collected from the cauda epididymis and fixed with glutaraldehyde. B and C ) Sperm collected from the cauda epididymis extracted with 0.1% Triton X-100 for 5 min before fixation in glutaraldehyde.
    Figure Legend Snippet: SEM of sperm from Eno4 Gt/Gt and WT mice. A and D ) Sperm collected from the cauda epididymis and fixed with glutaraldehyde. B and C ) Sperm collected from the cauda epididymis extracted with 0.1% Triton X-100 for 5 min before fixation in glutaraldehyde.

    Techniques Used: Mouse Assay

    A – C ) Expression of Eno1 transcripts and variants. A ) The location of primers specific for each of the Eno4 transcripts used in the PCR assays. B ) Conventional RT-PCR results with specific primer pairs detecting Eno4 transcript and variants in
    Figure Legend Snippet: A – C ) Expression of Eno1 transcripts and variants. A ) The location of primers specific for each of the Eno4 transcripts used in the PCR assays. B ) Conventional RT-PCR results with specific primer pairs detecting Eno4 transcript and variants in

    Techniques Used: Expressing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    ENO4 Is Required for Male Fertility
    Figure Legend Snippet: ENO4 Is Required for Male Fertility

    Techniques Used:

    TEM of sperm from Eno4 Gt/Gt and WT mice. Sperm collected from cauda epididymis were fixed, sectioned, and examined by TEM. Cross sections ( A – C ) and sagittal section ( D ) of sperm flagellum are shown. A , B , and D ) Sperm from Eno4 Gt/Gt mouse. C )
    Figure Legend Snippet: TEM of sperm from Eno4 Gt/Gt and WT mice. Sperm collected from cauda epididymis were fixed, sectioned, and examined by TEM. Cross sections ( A – C ) and sagittal section ( D ) of sperm flagellum are shown. A , B , and D ) Sperm from Eno4 Gt/Gt mouse. C )

    Techniques Used: Transmission Electron Microscopy, Mouse Assay

    Differences in solubility of PGAM2, enolase, ENO4, and AKAP4. Most of the PGAM2 (detected with PGAM antibody) and some of the enolase (detected with pan-enolase antibody) was detected in the 1% NP-40 sperm lysate. The majority of the enolase was solubilized
    Figure Legend Snippet: Differences in solubility of PGAM2, enolase, ENO4, and AKAP4. Most of the PGAM2 (detected with PGAM antibody) and some of the enolase (detected with pan-enolase antibody) was detected in the 1% NP-40 sperm lysate. The majority of the enolase was solubilized

    Techniques Used: Solubility

    25) Product Images from "The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription"

    Article Title: The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl181

    Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.
    Figure Legend Snippet: Effect of DmTTF-targeted RNAi in D.Mel-2 cells. ( A ) Western blotting analysis of D.Mel-2 total cell and mitochondrial lysate. A total of 250 µg of proteins were fractionated on a 12% SDS–polyacrylamide gel, electroblotted to PVDF filters and incubated with polyclonal antibodies against recombinant DmTTF. ( B ) D.Mel-2 cells were either untreated (control) or treated with odds-paired (Opa1) or DmTTF dsRNA. Mitochondrial lysate (250 µg of proteins) was probed with polyclonal antibodies against DmTTF, h-NDUFS4 or D-TFAM.

    Techniques Used: Western Blot, Incubation, Recombinant

    26) Product Images from "Effect of Levels of Acetate on the Mevalonate Pathway of Borrelia burgdorferi"

    Article Title: Effect of Levels of Acetate on the Mevalonate Pathway of Borrelia burgdorferi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038171

    Sensitivity of the HMGR overexpression strain, TR1, to statins. Equivalent numbers of spirochetes from B. burgdorferi ML23 or TR1 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) treated with 5 mM IPTG for 16 hrs to induce HMGR expression as described under Materials and Methods and were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblot (B) was probed with anti-serum against HMGR. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons. (C) Spirochetes were analyzed for sensitivity to increasing concentrations of stains. Data shown are the average of three independent assays; bars indicate the standard deviation. Data were subjected to the unpaired Student's t test implemented in Excel software. Asterisks indicate samples whose values are statistically significantly different between control and overexpression strains (**, P
    Figure Legend Snippet: Sensitivity of the HMGR overexpression strain, TR1, to statins. Equivalent numbers of spirochetes from B. burgdorferi ML23 or TR1 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) treated with 5 mM IPTG for 16 hrs to induce HMGR expression as described under Materials and Methods and were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblot (B) was probed with anti-serum against HMGR. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons. (C) Spirochetes were analyzed for sensitivity to increasing concentrations of stains. Data shown are the average of three independent assays; bars indicate the standard deviation. Data were subjected to the unpaired Student's t test implemented in Excel software. Asterisks indicate samples whose values are statistically significantly different between control and overexpression strains (**, P

    Techniques Used: Over Expression, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Standard Deviation, Software

    Levels of proteins of the MP in B. burgdorferi . Equivalent number of spirochetes from B. burgdorferi strain B31-A3 propagated in BSK-II medium with 6% NRS under conditions that either mimicked the unfed-tick (pH 7.6/23°C; Lane 1) or fed-tick (pH 6.8/37°C; Lane 2) to a density of 5 × 10 7 spirochetes/ml were resolved by SDS-12.5%PAGE. Gels were stained with Coomassie blue (A) or separated proteins were electrotransfered onto PVDF membranes. (B) Immunoblots were incubated with mouse serum against purified HMGR, MvaD, Pmk, Mvk and P66 respectively. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Higher levels of HMGR expression were seen under fed-tick conditions when compared to unfed-tick conditions while the other three proteins showed higher levels of expression under unfed-tick conditions when compared to fed-tick conditions.
    Figure Legend Snippet: Levels of proteins of the MP in B. burgdorferi . Equivalent number of spirochetes from B. burgdorferi strain B31-A3 propagated in BSK-II medium with 6% NRS under conditions that either mimicked the unfed-tick (pH 7.6/23°C; Lane 1) or fed-tick (pH 6.8/37°C; Lane 2) to a density of 5 × 10 7 spirochetes/ml were resolved by SDS-12.5%PAGE. Gels were stained with Coomassie blue (A) or separated proteins were electrotransfered onto PVDF membranes. (B) Immunoblots were incubated with mouse serum against purified HMGR, MvaD, Pmk, Mvk and P66 respectively. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Higher levels of HMGR expression were seen under fed-tick conditions when compared to unfed-tick conditions while the other three proteins showed higher levels of expression under unfed-tick conditions when compared to fed-tick conditions.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Incubation, Purification, Expressing

    Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under conditions that mimic unfed ticks. Equivalent numbers of spirochetes from A3 propagated in BSK-II medium with 6% NRS under conditions that mimicked the unfed-tick (pH 7.6/23°C) with increasing concentrations of supplemental NaOAc (from 0 mM –90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels of protein expression were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA4, OppA5 (C), gene regulators RpoS and CsrA Bb (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F). Decreased levels of protein expression were seen with Pta (E). Acetate levels had no effect on the other proteins tested.
    Figure Legend Snippet: Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under conditions that mimic unfed ticks. Equivalent numbers of spirochetes from A3 propagated in BSK-II medium with 6% NRS under conditions that mimicked the unfed-tick (pH 7.6/23°C) with increasing concentrations of supplemental NaOAc (from 0 mM –90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels of protein expression were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA4, OppA5 (C), gene regulators RpoS and CsrA Bb (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F). Decreased levels of protein expression were seen with Pta (E). Acetate levels had no effect on the other proteins tested.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Expressing

    Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under laboratory growth conditions. Equivalent numbers of spirochetes from B. burgdorferi B31-A3 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) with increasing concentrations of supplemental NaOAc (from 0 mM –90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels of protein expression were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA5 (C); gene regulators RpoS, CsrA Bb , and BosR (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F); NapA (G). Decreased levels of protein expression were seen with Pta (E). Acetate levels had no effect on the other proteins tested.
    Figure Legend Snippet: Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under laboratory growth conditions. Equivalent numbers of spirochetes from B. burgdorferi B31-A3 propagated in BSK-II medium with 6% NRS at laboratory growth conditions (pH 7.6/32°C) with increasing concentrations of supplemental NaOAc (from 0 mM –90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. Numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels of protein expression were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA5 (C); gene regulators RpoS, CsrA Bb , and BosR (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F); NapA (G). Decreased levels of protein expression were seen with Pta (E). Acetate levels had no effect on the other proteins tested.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Expressing

    Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under conditions that mimic fed-ticks. Equivalent numbers of spirochetes from B. burgdorferi B31-A3 propagated in BSK-II medium with 6% NRS under conditions that mimicked the fed-tick (pH 6.8/37°C) with increasing concentrations of supplemental NaOAc (from 0 mM - 90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. The numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA5 (C); gene regulators RpoS and CsrA Bb (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F). Decreased levels of protein expression were seen with OppA2 (C), Pta (E), and FlaB (G). Acetate levels had no effect on the other proteins tested.
    Figure Legend Snippet: Effect of increasing concentrations of sodium acetate on levels of borrelial proteins under conditions that mimic fed-ticks. Equivalent numbers of spirochetes from B. burgdorferi B31-A3 propagated in BSK-II medium with 6% NRS under conditions that mimicked the fed-tick (pH 6.8/37°C) with increasing concentrations of supplemental NaOAc (from 0 mM - 90 mM) were resolved by SDS-12.5%PAGE. The gels were stained with Coomassie blue (A) or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots (B-G) were probed with anti-serum against the antigens listed to the right of the blots. Blots were developed using the Enhanced Chemiluminescence system. The numbers to the left of the panels indicate the molecular mass standards in kilodaltons proximate to each of the antigens. Increased levels were seen with increasing concentrations of NaOAc for all proteins of the mevalonate pathway (B); OppA5 (C); gene regulators RpoS and CsrA Bb (D); AckA (E); pathogenesis-related proteins DbpA, BBK32, and OspC (F). Decreased levels of protein expression were seen with OppA2 (C), Pta (E), and FlaB (G). Acetate levels had no effect on the other proteins tested.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Expressing

    27) Product Images from "CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi ▿"

    Article Title: CsrA Modulates Levels of Lipoproteins and Key Regulators of Gene Expression Critical for Pathogenic Mechanisms of Borrelia burgdorferi ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00882-10

    Deletion of csrA Bb results in decreased expression of borrelial determinants associated with pathogenesis. Equivalent numbers of spirochetes from ML23 (wt) and ES10 (mt) propagated in BSK-II medium with 6% normal rabbit serum to a density of 5 × 10 7 spirochetes/ml under conditions that mimicked either the unfed tick (pH 7.6/23°C; lane 1) or fed tick (pH 6.8/37°C; lane 2) were resolved by SDS-12.5% PAGE. The gels were stained with Coomassie blue (A), or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots were developed with antibodies consisting of mouse or rabbit serum against regulators of gene expression indicated to the right (B) or with antibodies consisting of mouse, rat, or rabbit antisera against the proteins indicated to the right (C). The blots were developed using an enhanced chemiluminescence system. The asterisk in panel A indicates increased levels of OspC in the wt strain under the fed-tick condition. The numbers to the left of the panels indicate the molecular mass standards (in kilodaltons) proximate to each of the antigens.
    Figure Legend Snippet: Deletion of csrA Bb results in decreased expression of borrelial determinants associated with pathogenesis. Equivalent numbers of spirochetes from ML23 (wt) and ES10 (mt) propagated in BSK-II medium with 6% normal rabbit serum to a density of 5 × 10 7 spirochetes/ml under conditions that mimicked either the unfed tick (pH 7.6/23°C; lane 1) or fed tick (pH 6.8/37°C; lane 2) were resolved by SDS-12.5% PAGE. The gels were stained with Coomassie blue (A), or the separated proteins were electrotransfered onto PVDF membranes. Immunoblots were developed with antibodies consisting of mouse or rabbit serum against regulators of gene expression indicated to the right (B) or with antibodies consisting of mouse, rat, or rabbit antisera against the proteins indicated to the right (C). The blots were developed using an enhanced chemiluminescence system. The asterisk in panel A indicates increased levels of OspC in the wt strain under the fed-tick condition. The numbers to the left of the panels indicate the molecular mass standards (in kilodaltons) proximate to each of the antigens.

    Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, Staining, Western Blot

    28) Product Images from "Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38"

    Article Title: Cellular N-Ras Promotes Cell Survival by Downregulation of Jun N-Terminal Protein Kinase and p38

    Journal: Molecular and Cellular Biology

    doi:

    Expression of switch 1 effector domain mutant N-Ras constructs in the N-Ras knockout cells and NIH 3T3 cells. (A) N-Ras knockout cells were transfected with three different switch 1 mutant N-Ras constructs (K61:35S, K61:37G, and K61:40C) and stable clones were selected in G418 as described in Materials and Methods. A 100-μg portion of each lysate (including the parental N-Ras knockout cells and control N +/+ cells) was electrophoresed on SDS-13% polyacrylamide gels and transferred to PVDF. The blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Detection was performed by standard ECL techniques. (B) N-Ras knockout and control N +/+ cells and N-Ras knockout cells stably expressing the switch 1 mutant N-Ras proteins were subcultured in serum-containing medium and left untreated. At 48 h after passage, the cells were harvested and lysates were prepared as described in Materials and Methods. A 20-μg portion of each lysate was electrophoresed on two separate 13% polyacrylamide gels and transferred to PVDF. The upper blot was developed with anti-phospho-Erk (pErk) and anti-mouse antibody-HRP, and the lower blot was developed with anti-Erk2 polyclonal antibody and anti-rabbit-HRP for total Erk2. Bands were visualized by standard ECL techniques. (C), NIH 3T3 cells were transfected with the indicated constructs, including the effector domain mutant N-Ras constructs, as described in Materials and Methods. (Upper panel) Expression levels of ectopic N-Ras proteins in NIH 3T3 cells was determined by Western analysis. Cultures were grown to approximately 80% confluency and then lysed in a detergent buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM NaVO 3 , 1 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride, 1 μg of aprotinin per ml, 1 μg of leupeptin per ml). Protein concentrations were determined with the bicinchoninic acid kit (Pierce). SDS-PAGE was used to resolve 40 μg of total protein from each sample on a 15% polyacrylamide gel. Proteins were transferred to Immobilon-P membranes (Millipore), and the blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Bands were visualized by standard ECL (Amersham) techniques. (Lower panel) Lysates prepared as described for the upper panel were used to determine the steady-state levels of Erk expression and activation. Following electrophoresis and transfer, the blot was developed with phospho-p44/42 MAP kinase (T202/Y204) E10 antibody (1:1,000; Cell Signaling Technology). Detection was performed by ECL (Amersham) following incubation with HRP-conjugated secondary anti-mouse IgG antibodies. (D) N-Ras knockout and control N +/+ cells and N-Ras knockout cells expressing the switch 1 effector domain mutant N-Ras constructs were grown to approximately 70% confluence in complete serum-containing medium for 2 days. The cells were harvested, and lysates were prepared in the lysis buffer supplied by the manufacturer. Protein concentrations were determined as described in Materials and Methods. Immobilized Akt 1G1 monoclonal antibody (20 μl of slurry) was used to immunoprecipitate Akt from 0.5 mg of each lysate for 3 h at 4°C. The immunocomplexes were washed and used in a kinase assay as specified by the manufacturer, except that 2 μg of the substrate GSK-3 fusion protein was used per reaction. The supernatants were removed, the pellets were washed with 30 μl of buffer, and the supernatants were combined and electrophoresed on SDS-12% polyacrylamide gels. Following transfer to PVDF, the blot was developed with anti-phsopho-GSK3α/β (pGSK-3β) polyclonal antibody and goat-anti-rabbit-HRP secondary antibody followed by donkey anti-goat antibody-HRP tertiary amplification. Detection was performed using standard ECL techniques.
    Figure Legend Snippet: Expression of switch 1 effector domain mutant N-Ras constructs in the N-Ras knockout cells and NIH 3T3 cells. (A) N-Ras knockout cells were transfected with three different switch 1 mutant N-Ras constructs (K61:35S, K61:37G, and K61:40C) and stable clones were selected in G418 as described in Materials and Methods. A 100-μg portion of each lysate (including the parental N-Ras knockout cells and control N +/+ cells) was electrophoresed on SDS-13% polyacrylamide gels and transferred to PVDF. The blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Detection was performed by standard ECL techniques. (B) N-Ras knockout and control N +/+ cells and N-Ras knockout cells stably expressing the switch 1 mutant N-Ras proteins were subcultured in serum-containing medium and left untreated. At 48 h after passage, the cells were harvested and lysates were prepared as described in Materials and Methods. A 20-μg portion of each lysate was electrophoresed on two separate 13% polyacrylamide gels and transferred to PVDF. The upper blot was developed with anti-phospho-Erk (pErk) and anti-mouse antibody-HRP, and the lower blot was developed with anti-Erk2 polyclonal antibody and anti-rabbit-HRP for total Erk2. Bands were visualized by standard ECL techniques. (C), NIH 3T3 cells were transfected with the indicated constructs, including the effector domain mutant N-Ras constructs, as described in Materials and Methods. (Upper panel) Expression levels of ectopic N-Ras proteins in NIH 3T3 cells was determined by Western analysis. Cultures were grown to approximately 80% confluency and then lysed in a detergent buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM NaVO 3 , 1 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride, 1 μg of aprotinin per ml, 1 μg of leupeptin per ml). Protein concentrations were determined with the bicinchoninic acid kit (Pierce). SDS-PAGE was used to resolve 40 μg of total protein from each sample on a 15% polyacrylamide gel. Proteins were transferred to Immobilon-P membranes (Millipore), and the blot was developed with anti-N-Ras monoclonal antibody and anti-mouse secondary antibody-HRP. Bands were visualized by standard ECL (Amersham) techniques. (Lower panel) Lysates prepared as described for the upper panel were used to determine the steady-state levels of Erk expression and activation. Following electrophoresis and transfer, the blot was developed with phospho-p44/42 MAP kinase (T202/Y204) E10 antibody (1:1,000; Cell Signaling Technology). Detection was performed by ECL (Amersham) following incubation with HRP-conjugated secondary anti-mouse IgG antibodies. (D) N-Ras knockout and control N +/+ cells and N-Ras knockout cells expressing the switch 1 effector domain mutant N-Ras constructs were grown to approximately 70% confluence in complete serum-containing medium for 2 days. The cells were harvested, and lysates were prepared in the lysis buffer supplied by the manufacturer. Protein concentrations were determined as described in Materials and Methods. Immobilized Akt 1G1 monoclonal antibody (20 μl of slurry) was used to immunoprecipitate Akt from 0.5 mg of each lysate for 3 h at 4°C. The immunocomplexes were washed and used in a kinase assay as specified by the manufacturer, except that 2 μg of the substrate GSK-3 fusion protein was used per reaction. The supernatants were removed, the pellets were washed with 30 μl of buffer, and the supernatants were combined and electrophoresed on SDS-12% polyacrylamide gels. Following transfer to PVDF, the blot was developed with anti-phsopho-GSK3α/β (pGSK-3β) polyclonal antibody and goat-anti-rabbit-HRP secondary antibody followed by donkey anti-goat antibody-HRP tertiary amplification. Detection was performed using standard ECL techniques.

    Techniques Used: Expressing, Mutagenesis, Construct, Knock-Out, Transfection, Clone Assay, Stable Transfection, Western Blot, SDS Page, Activation Assay, Electrophoresis, Incubation, Lysis, Kinase Assay, Amplification

    c-N-Ras downregulates JNK activation and activity following treatment with apoptotic agonists. (A) N-Ras knockout fibroblasts, control N +/+ cells, and c-N-Ras reconstituted cells were left untreated ( t = 0) or treated for various times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of three different experiments. (B) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were untreated or treated with TNF-α and cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-JNK1 rabbit polyclonal antibody and anti-rabbit secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of two different experiments. (C) N-Ras knockout cells, control N +/+ cells and c-N-Ras reconstituted cells [cell lines N −/− (2)/wtN3 and N −/− (2)/wtN8] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. JNK1 was immunoprecipitated from 50 μg of each lysate. The immunocomplexes were incubated for 20 min at 37°C in kinase buffer. The supernatants, which contain the ATF2 substrate, were loaded onto SDS-12% polyacrylamide gels, electrophoresed, and transferred to PVDF. Signals were detected using a PhosphorImager. A nonimmune (NI) control was performed using an equal amount (1.5 μg) of purified rabbit (Rb) IgG and the 1-h TNF-α-plus-cycloheximide-treated N +/+ (1) cell lysate. The results are representative of three separate experiments. (D) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)wtN3] were left untreated or treated with 1 μg of activating anti-Fas antibody per ml in the presence of 0.5 μg of protein G per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of each lysate was electrophoresed on an SDS-10% polyacrylamide gel and transferred to PVDF. The blot was developed with anti-pJNK monoclonal antibody and goat anti-mouse IgG-HRP as described for panel A. Detection was performed using standard ECL techniques. The positive control was N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of six separate experiments. (E) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with anti-Fas antibody and protein G as described for panel D. At the indicated times, the cells were harvested and lysates were prepared for immunoprecipitation of JNK as described for panel C. The immunoprecipitated JNK from each sample was used in a kinase reaction as described for panel C, using GST-ATF2 as the substrate. Following electrophoresis and transfer to PVDF, the radioactivity incorporated into the GST-ATF2 substrate was detected using a PhosphorImager. The nonimmune control (NI) used rabbit IgG and the N +/+ (1) cells treated for 1 h, as described for panel C. The results are representative of two separate experiments. (F) N-Ras knockout and control cells and N-Ras knockout cells ectopically overexpressing c-K(A)-Ras (clones 4 and 8) were left untreated or treated for 1 or 4 h with TNF-α plus cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse-HRP secondary antibody. Detection was performed using standard ECL techniques.
    Figure Legend Snippet: c-N-Ras downregulates JNK activation and activity following treatment with apoptotic agonists. (A) N-Ras knockout fibroblasts, control N +/+ cells, and c-N-Ras reconstituted cells were left untreated ( t = 0) or treated for various times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of three different experiments. (B) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were untreated or treated with TNF-α and cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-JNK1 rabbit polyclonal antibody and anti-rabbit secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of two different experiments. (C) N-Ras knockout cells, control N +/+ cells and c-N-Ras reconstituted cells [cell lines N −/− (2)/wtN3 and N −/− (2)/wtN8] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. JNK1 was immunoprecipitated from 50 μg of each lysate. The immunocomplexes were incubated for 20 min at 37°C in kinase buffer. The supernatants, which contain the ATF2 substrate, were loaded onto SDS-12% polyacrylamide gels, electrophoresed, and transferred to PVDF. Signals were detected using a PhosphorImager. A nonimmune (NI) control was performed using an equal amount (1.5 μg) of purified rabbit (Rb) IgG and the 1-h TNF-α-plus-cycloheximide-treated N +/+ (1) cell lysate. The results are representative of three separate experiments. (D) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)wtN3] were left untreated or treated with 1 μg of activating anti-Fas antibody per ml in the presence of 0.5 μg of protein G per ml. At the indicated times, the cells were harvested and lysates were prepared as described in the text. A 50-μg portion of each lysate was electrophoresed on an SDS-10% polyacrylamide gel and transferred to PVDF. The blot was developed with anti-pJNK monoclonal antibody and goat anti-mouse IgG-HRP as described for panel A. Detection was performed using standard ECL techniques. The positive control was N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of six separate experiments. (E) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with anti-Fas antibody and protein G as described for panel D. At the indicated times, the cells were harvested and lysates were prepared for immunoprecipitation of JNK as described for panel C. The immunoprecipitated JNK from each sample was used in a kinase reaction as described for panel C, using GST-ATF2 as the substrate. Following electrophoresis and transfer to PVDF, the radioactivity incorporated into the GST-ATF2 substrate was detected using a PhosphorImager. The nonimmune control (NI) used rabbit IgG and the N +/+ (1) cells treated for 1 h, as described for panel C. The results are representative of two separate experiments. (F) N-Ras knockout and control cells and N-Ras knockout cells ectopically overexpressing c-K(A)-Ras (clones 4 and 8) were left untreated or treated for 1 or 4 h with TNF-α plus cycloheximide as described for panel A. A 50-μg portion of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer, the blot was developed with anti-phospho-JNK (pJNK) and anti-mouse-HRP secondary antibody. Detection was performed using standard ECL techniques.

    Techniques Used: Activation Assay, Activity Assay, Knock-Out, Electrophoresis, Immunoprecipitation, Incubation, Purification, Positive Control, Radioactivity

    c-N-Ras downregulates p38 activation and activity following the induction of apoptosis. (A) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN43A] were left untreated or treated for varying times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. A 50-μg portion of each lysate was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer, the blot was developed with anti-phospho-p38 (pp38) and anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to immunoprecipitate p38 as described in Materials and Methods. The resulting immunocomplexes were washed and used in a kinase assay as described in the text with 25 μCi of [γ- 32 P]ATP per reaction and GST-ATF2 as the substrate. Signals were detected with a PhosphorImager. The nonimmune control (NI) consisted of immunoprecipitation of 1 h treated N +/+ (1) control cells using an equal amount of nonimmune rabbit serum. The results are representative of four separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to analyze total levels of p38 using anti-p38 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The results are representative of two separate experiments. (D) N-Ras knockout cells [N −/− (3) cell line], control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (3)/wtN5] were left untreated in complete medium or starved of serum for various times as described in Materials and Methods. At the indicated times, cell lysates were prepared. A 50-μg portion of each lysates was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-p38 and anti-rabbit secondary antibody-HRP as described in the text. Bands were visualized by standard ECL techniques. The results are representative of two separate experiments.
    Figure Legend Snippet: c-N-Ras downregulates p38 activation and activity following the induction of apoptosis. (A) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN43A] were left untreated or treated for varying times with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, the cells were harvested and lysates prepared as described in the text. A 50-μg portion of each lysate was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer, the blot was developed with anti-phospho-p38 (pp38) and anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) N-Ras knockout and control cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to immunoprecipitate p38 as described in Materials and Methods. The resulting immunocomplexes were washed and used in a kinase assay as described in the text with 25 μCi of [γ- 32 P]ATP per reaction and GST-ATF2 as the substrate. Signals were detected with a PhosphorImager. The nonimmune control (NI) consisted of immunoprecipitation of 1 h treated N +/+ (1) control cells using an equal amount of nonimmune rabbit serum. The results are representative of four separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. At the indicated times, cell lysates were prepared and 50 μg of each lysate was used to analyze total levels of p38 using anti-p38 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. The results are representative of two separate experiments. (D) N-Ras knockout cells [N −/− (3) cell line], control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (3)/wtN5] were left untreated in complete medium or starved of serum for various times as described in Materials and Methods. At the indicated times, cell lysates were prepared. A 50-μg portion of each lysates was loaded onto an SDS-10% polyacrylamide gel, and following electrophoresis and transfer to PVDF, the blot was developed with anti-phospho-p38 and anti-rabbit secondary antibody-HRP as described in the text. Bands were visualized by standard ECL techniques. The results are representative of two separate experiments.

    Techniques Used: Activation Assay, Activity Assay, Knock-Out, Electrophoresis, Kinase Assay, Immunoprecipitation

    c-N-Ras downregulates the upstream kinase activators of JNK and p38. (A) (Upper panel) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN3] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 100 μg of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer to PVDF, the blot was incubated with anti-phospho-MKK4 (pMKK4) polyclonal antibody. The blot was developed by using anti-rabbit secondar y antibody-HRP and standard ECL techniques. The positive control consisted of N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of four separate experiments. (Lower panel) A 100-μg portion of the same lysates as in the upper panel was electrophoresed, transferred, and blotted with anti-MKK4 polyclonal antibody (1:1,000 dilution) followed by anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) (Upper panel) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 100 μg of protein of each lysate was electrophoresed and transferred to PVDF. The blot was developed with anti-phospo-MKK3/6 (pMKK3/6) antibody and anti-rabbit secondary antibody-HRP. Bands were visualized by standard ECL techniques. This experiment is representative of three separate experiments. (Lower panel) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A. Cell lysates (100 μg) were prepared for analysis of total MKK3 levels by using anti-MKK3 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. (C) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. Cell lysates were prepared at the indicated times, and protein concentrations determined. A 2-μg portion of unactive His 6 -tagged JNK1α1 was incubated with 12 μg of each lysate in the presence of 50 μM unlabeled ATP for 20 min at 30°C. The added His 6 -JNK1α1 was isolated by incubation with nickel resin (ProBond; Invitrogen) for 45 min at 4°C. The resin was washed with p21 buffer and used in a second kinase assay to measure the activity of the isolated, recombinant JNK1α1 by using GST-ATF2 as the substrate as described in Materials and Methods. The unactive JNK1α1 incubated in the absence of lysate had no activity. The results are representative of three separate experiments. (D) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A, and at the indicated times cell lysates were prepared. A 1-μg portion of unactive GST-p38α was incubated for 20 min at 30°C, as described in the text, with 12 μg of each lysate in p21 buffer containing 50 μM ATP. The GST-p38α was isolated with glutathione-agarose, and its activity was measured in a second kinase reaction using [γ- 32 P]ATP with GST-ATF2 as the substrate, as described in Materials and Methods. N-Ras knockout cells were treated for 1 h with 20 μg of anisomycin per ml, and the lysate was used to activate the GST-p38α as a positive control. The unactive GST-p38α had no activity when incubated in the absence of cell lysate. (E) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were passaged into complete medium and 48 h later were either left untreated ( t = 0) or challenged with 10 ng of EGF per ml for the times indicated. Cell lysates prepared at the indicated times were electrophoresed and transferred to PVDF. The membranes were immunoblotted for phospho-JNK (upper panel, 50 μg/lane) and pErk (Santa Cruz Biotechnology; lower panel, 20 μg/lane). The results are representative of three separate experiments.
    Figure Legend Snippet: c-N-Ras downregulates the upstream kinase activators of JNK and p38. (A) (Upper panel) N-Ras knockout cells, control N +/+ cells, and c-N-Ras reconstituted cells [N −/− (2)/wtN3] were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 100 μg of protein was loaded in each lane of an SDS-10% polyacrylamide gel. Following electrophoresis and transfer to PVDF, the blot was incubated with anti-phospho-MKK4 (pMKK4) polyclonal antibody. The blot was developed by using anti-rabbit secondar y antibody-HRP and standard ECL techniques. The positive control consisted of N-Ras knockout cells treated for 1 h with 20 μg of anisomycin (Aniso) per ml. The results are representative of four separate experiments. (Lower panel) A 100-μg portion of the same lysates as in the upper panel was electrophoresed, transferred, and blotted with anti-MKK4 polyclonal antibody (1:1,000 dilution) followed by anti-rabbit secondary antibody-HRP. Detection was performed by standard ECL techniques. The results are representative of two separate experiments. (B) (Upper panel) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as in panel A. At the indicated times, cell lysates were prepared and 100 μg of protein of each lysate was electrophoresed and transferred to PVDF. The blot was developed with anti-phospo-MKK3/6 (pMKK3/6) antibody and anti-rabbit secondary antibody-HRP. Bands were visualized by standard ECL techniques. This experiment is representative of three separate experiments. (Lower panel) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A. Cell lysates (100 μg) were prepared for analysis of total MKK3 levels by using anti-MKK3 polyclonal antibody. The blot was developed by using anti-rabbit secondary antibody-HRP and standard ECL techniques. (C) N-Ras knockout and control N +/+ cells and c-N-Ras reconstituted cells were left untreated or treated with TNF-α and cycloheximide as described for panel A. Cell lysates were prepared at the indicated times, and protein concentrations determined. A 2-μg portion of unactive His 6 -tagged JNK1α1 was incubated with 12 μg of each lysate in the presence of 50 μM unlabeled ATP for 20 min at 30°C. The added His 6 -JNK1α1 was isolated by incubation with nickel resin (ProBond; Invitrogen) for 45 min at 4°C. The resin was washed with p21 buffer and used in a second kinase assay to measure the activity of the isolated, recombinant JNK1α1 by using GST-ATF2 as the substrate as described in Materials and Methods. The unactive JNK1α1 incubated in the absence of lysate had no activity. The results are representative of three separate experiments. (D) Cells were left untreated or treated with TNF-α and cycloheximide as in panel A, and at the indicated times cell lysates were prepared. A 1-μg portion of unactive GST-p38α was incubated for 20 min at 30°C, as described in the text, with 12 μg of each lysate in p21 buffer containing 50 μM ATP. The GST-p38α was isolated with glutathione-agarose, and its activity was measured in a second kinase reaction using [γ- 32 P]ATP with GST-ATF2 as the substrate, as described in Materials and Methods. N-Ras knockout cells were treated for 1 h with 20 μg of anisomycin per ml, and the lysate was used to activate the GST-p38α as a positive control. The unactive GST-p38α had no activity when incubated in the absence of cell lysate. (E) N-Ras knockout, control N +/+ , and c-N-Ras reconstituted cells were passaged into complete medium and 48 h later were either left untreated ( t = 0) or challenged with 10 ng of EGF per ml for the times indicated. Cell lysates prepared at the indicated times were electrophoresed and transferred to PVDF. The membranes were immunoblotted for phospho-JNK (upper panel, 50 μg/lane) and pErk (Santa Cruz Biotechnology; lower panel, 20 μg/lane). The results are representative of three separate experiments.

    Techniques Used: Knock-Out, Electrophoresis, Incubation, Positive Control, Isolation, Kinase Assay, Activity Assay, Recombinant

    29) Product Images from "Chromosomal localization links the SIN3-RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression"

    Article Title: Chromosomal localization links the SIN3-RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.22.6131

    Fig. 1. SIN3 and RPD3 polyclonal antibodies are highly specific. ( A ) Schematic diagrams of SIN3 and RPD3 proteins. Solid bars indicate regions used as antigens for generating polyclonal antibodies. The RPD3 region does not include the deacetylase domain, indicated by a shaded box. The SIN3 region contains paired-amphipathic helix (PAH) 4, indicated by a solid box, and a conserved region of undefined function, indicated by a hatched box, but does not include PAH1–3 or the histone deacetylase interaction domain (HID). ( B ) Western blots of total protein extracted from 0–12 h Drosophila embryos (Emb.) and from larval salivary glands (S.G.) were probed with purified RPD3 antibody (lanes 1 and 2) or SIN3 antibody (lanes 3 and 4). The positions of protein molecular weight size markers are indicated on the left.
    Figure Legend Snippet: Fig. 1. SIN3 and RPD3 polyclonal antibodies are highly specific. ( A ) Schematic diagrams of SIN3 and RPD3 proteins. Solid bars indicate regions used as antigens for generating polyclonal antibodies. The RPD3 region does not include the deacetylase domain, indicated by a shaded box. The SIN3 region contains paired-amphipathic helix (PAH) 4, indicated by a solid box, and a conserved region of undefined function, indicated by a hatched box, but does not include PAH1–3 or the histone deacetylase interaction domain (HID). ( B ) Western blots of total protein extracted from 0–12 h Drosophila embryos (Emb.) and from larval salivary glands (S.G.) were probed with purified RPD3 antibody (lanes 1 and 2) or SIN3 antibody (lanes 3 and 4). The positions of protein molecular weight size markers are indicated on the left.

    Techniques Used: Histone Deacetylase Assay, Western Blot, Purification, Molecular Weight

    Fig. 2. SIN3 and RPD3 co-localize throughout euchromatin but are absent from heterochromatin. ( A – E ) A single polytene chromosome spread stained for both SIN3 and RPD3 and counterstained with DAPI. In (E), the diamond and sphere indicate loci that stain predominantly for SIN3 and RPD3, respectively. ( F ) Higher magnification image of a spread co-stained for SIN3 and RPD3. ( G – I ) Higher magnification images of another spread stained for SIN3 and counterstained with DAPI. Arrows highlight the non-overlapping pattern of SIN3 and DAPI. Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (X, 2L, 2R, 3L, 3R and 4) are indicated at the tip, and the chromocenter is indicated by ‘C’. In (A–E), the chromocenter is broken into two pieces. Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence.
    Figure Legend Snippet: Fig. 2. SIN3 and RPD3 co-localize throughout euchromatin but are absent from heterochromatin. ( A – E ) A single polytene chromosome spread stained for both SIN3 and RPD3 and counterstained with DAPI. In (E), the diamond and sphere indicate loci that stain predominantly for SIN3 and RPD3, respectively. ( F ) Higher magnification image of a spread co-stained for SIN3 and RPD3. ( G – I ) Higher magnification images of another spread stained for SIN3 and counterstained with DAPI. Arrows highlight the non-overlapping pattern of SIN3 and DAPI. Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (X, 2L, 2R, 3L, 3R and 4) are indicated at the tip, and the chromocenter is indicated by ‘C’. In (A–E), the chromocenter is broken into two pieces. Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence.

    Techniques Used: Staining, Fluorescence

    Fig. 5. SIN3 binds steroid hormone-regulated loci. ( A ) A single polytene chromosome spread stained for both SIN3 and SMRTER. ( B – D ) Higher magnification images of a section of the spread in (A). Spheres indicate loci strongly stained for both SIN3 and SMRTER (yellow); a diamond indicates a locus stained for SIN3 but not SMRTER (green). Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (X, 2L, 2R, 3L, 3R and 4) are indicated at the tip and the chromocenter is indicated by a C. Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence. ( E and F ) High magnification images of chromosome 4 of spreads stained with SIN3 prepared from flies of a wild-type (E) or an Sgs-4 transgenic (F) line. The transgenic line has a fluorescent signal at the P-element insertion site at 102D3-5 (yellow arrow) in addition to the single strong band present in the wild-type line (red arrow).
    Figure Legend Snippet: Fig. 5. SIN3 binds steroid hormone-regulated loci. ( A ) A single polytene chromosome spread stained for both SIN3 and SMRTER. ( B – D ) Higher magnification images of a section of the spread in (A). Spheres indicate loci strongly stained for both SIN3 and SMRTER (yellow); a diamond indicates a locus stained for SIN3 but not SMRTER (green). Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (X, 2L, 2R, 3L, 3R and 4) are indicated at the tip and the chromocenter is indicated by a C. Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence. ( E and F ) High magnification images of chromosome 4 of spreads stained with SIN3 prepared from flies of a wild-type (E) or an Sgs-4 transgenic (F) line. The transgenic line has a fluorescent signal at the P-element insertion site at 102D3-5 (yellow arrow) in addition to the single strong band present in the wild-type line (red arrow).

    Techniques Used: Staining, Fluorescence, Transgenic Assay

    Fig. 6. SIN3 binding changes during the transcription cycle. ( A , C , E , G and I ) Images of DAPI-stained polytene chromosome spreads. ( B , D , F , H and J . Panels are shown at the same magnification.
    Figure Legend Snippet: Fig. 6. SIN3 binding changes during the transcription cycle. ( A , C , E , G and I ) Images of DAPI-stained polytene chromosome spreads. ( B , D , F , H and J . Panels are shown at the same magnification.

    Techniques Used: Binding Assay, Staining

    Fig. 3. Chromosomal sites of SIN3 binding and histone hyperacetylation are mutually exclusive. ( A – E ) A section of a single polytene chromosome spread stained for both H4nonAc and SIN3. Horizontal arrows indicate a region with strong H4nonAc and SIN3 binding. The vertical arrow indicates binding of H4nonAc at the band–interband junction. ( F – J ) A section of a single polytene chromosome spread stained for both SIN3 and H4Ac8. In (A–J), spheres indicate loci that stain strongly with DAPI and α-H4Ac8, but do not stain with α-H4nonAc or α-SIN3. ( K – M ) Polytene chromosome spreads co-stained for SIN3 and H4Ac12, H4Ac5 or H3Ac9/14, respectively. Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (2L, 2R and 4) are indicated at the tip and the chromocenter is indicated by ‘C’ in (K). Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence.
    Figure Legend Snippet: Fig. 3. Chromosomal sites of SIN3 binding and histone hyperacetylation are mutually exclusive. ( A – E ) A section of a single polytene chromosome spread stained for both H4nonAc and SIN3. Horizontal arrows indicate a region with strong H4nonAc and SIN3 binding. The vertical arrow indicates binding of H4nonAc at the band–interband junction. ( F – J ) A section of a single polytene chromosome spread stained for both SIN3 and H4Ac8. In (A–J), spheres indicate loci that stain strongly with DAPI and α-H4Ac8, but do not stain with α-H4nonAc or α-SIN3. ( K – M ) Polytene chromosome spreads co-stained for SIN3 and H4Ac12, H4Ac5 or H3Ac9/14, respectively. Co-localization of two antibodies appears as yellow fluorescence. Chromosome arms (2L, 2R and 4) are indicated at the tip and the chromocenter is indicated by ‘C’ in (K). Antibodies used for staining are indicated at the bottom of each panel. The color of the lettering matches the color of the fluorescence.

    Techniques Used: Binding Assay, Staining, Fluorescence

    30) Product Images from "The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly"

    Article Title: The Cu chaperone CopZ is required for Cu homeostasis in Rhodobacter capsulatus and influences cytochrome cbb3 oxidase assembly

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14190

    Identification of a CopZ homologue in R. capsulatus . (A) Amino acid alignment of the R. capsulatus CopZ (R.c.) with the respective CopZ-homologues of Enterococcus hirae (E.h.) Bacillus subtilis (B.s.), Rhodobacter sphaeroides (R.s.), and Homo sapiens (H.s.). The conserved Cu-binding motif is shown in red. (B) Genetic organization of copZ in R. capsulatus . lepA and etp presumably encode for a translation factor and a phosphotyrosine protein phosphatase, respectively. The open reading frames rcc03124 and rcc03126 encode for hypothetical proteins. (C) R. capsulatus cells were grown in MPYE medium, precipitated with trichloroacetic acid and the pellet was dissolved loading buffer. After SDS-PAGE, the gel was either stained directly with coomassie brilliant blue (CBB) as loading control, or was blotted and decorated with α-CopZ antibodies (WB). WT corresponds to MT1131, Δ copZ to a MT1131 derivative carrying an insertion-deletion mutation within copZ and Δ copZ -pcopZ to the Δ copZ strain with a plasmid-encoded copZ . (D) MT1131 (WT) and Δ copZ cell extracts were separated into a soluble fraction and a membrane fraction by ultracentrifugation. Subsequently, the material was separated by SDS-PAGE and decorated with α-CopZ antibodies. (E) The cellular concentration of CopZ in MT1131 grown on MPYE medium without further Cu supplementation was determined by quantitative western blotting, using defined amounts of purified CopZ as reference. Signal intensity was quantified by ImageJ and several independent experiments were performed and a representative western blot is shown. Note that the purified CopZ contained a His-tag and it therefore migrates slower on SDS-PAGE than the native CopZ. (F) RT-PCR analyses of mRNA levels in wild type cells grown on MPYE without and with Cu supplementation (10 μM Cu(II)). A representative gel of three independent experiments is shown. The 16S ribosomal RNA served as control and the ccoI and copA mRNA as reference. Quantification was performed with ImageJ and signal intensity of the mRNA level in cells without Cu supplementation was set to 100%. (G) The CopZ levels in whole cells grown either on enriched medium (MPYE) or minimal medium (MedA) were analysed by immunoblotting as described above. When indicated, CuSO 4 was added to the growth medium. The levels of the Rieske Fe-S protein PetA served as loading control.
    Figure Legend Snippet: Identification of a CopZ homologue in R. capsulatus . (A) Amino acid alignment of the R. capsulatus CopZ (R.c.) with the respective CopZ-homologues of Enterococcus hirae (E.h.) Bacillus subtilis (B.s.), Rhodobacter sphaeroides (R.s.), and Homo sapiens (H.s.). The conserved Cu-binding motif is shown in red. (B) Genetic organization of copZ in R. capsulatus . lepA and etp presumably encode for a translation factor and a phosphotyrosine protein phosphatase, respectively. The open reading frames rcc03124 and rcc03126 encode for hypothetical proteins. (C) R. capsulatus cells were grown in MPYE medium, precipitated with trichloroacetic acid and the pellet was dissolved loading buffer. After SDS-PAGE, the gel was either stained directly with coomassie brilliant blue (CBB) as loading control, or was blotted and decorated with α-CopZ antibodies (WB). WT corresponds to MT1131, Δ copZ to a MT1131 derivative carrying an insertion-deletion mutation within copZ and Δ copZ -pcopZ to the Δ copZ strain with a plasmid-encoded copZ . (D) MT1131 (WT) and Δ copZ cell extracts were separated into a soluble fraction and a membrane fraction by ultracentrifugation. Subsequently, the material was separated by SDS-PAGE and decorated with α-CopZ antibodies. (E) The cellular concentration of CopZ in MT1131 grown on MPYE medium without further Cu supplementation was determined by quantitative western blotting, using defined amounts of purified CopZ as reference. Signal intensity was quantified by ImageJ and several independent experiments were performed and a representative western blot is shown. Note that the purified CopZ contained a His-tag and it therefore migrates slower on SDS-PAGE than the native CopZ. (F) RT-PCR analyses of mRNA levels in wild type cells grown on MPYE without and with Cu supplementation (10 μM Cu(II)). A representative gel of three independent experiments is shown. The 16S ribosomal RNA served as control and the ccoI and copA mRNA as reference. Quantification was performed with ImageJ and signal intensity of the mRNA level in cells without Cu supplementation was set to 100%. (G) The CopZ levels in whole cells grown either on enriched medium (MPYE) or minimal medium (MedA) were analysed by immunoblotting as described above. When indicated, CuSO 4 was added to the growth medium. The levels of the Rieske Fe-S protein PetA served as loading control.

    Techniques Used: Binding Assay, SDS Page, Staining, Western Blot, Mutagenesis, Plasmid Preparation, Concentration Assay, Purification, Reverse Transcription Polymerase Chain Reaction

    CopZ is a Cu binding protein that forms redox-sensitive oligomers. (A) Purified CopZ was separated on native, non-reducing PAGE and after western transfer was decorated with α-CopZ antibodies (left panel). The dominant CopZ species are indicated by numbers (1–3); two additional minor species are indicated by (*), but those were not further analyzed. The dominant species 1–3 were gel-extracted from the native gel and separated on a second dimension SDS-PAGE under reducing conditions (right panel). The second dimension was stained with coomassie brilliant blue. Indicated are CopZ and a second band (?) that was not characterized further. (B) Purified CopZ and its derivatives lacking either one (C10S) or both conserved cysteines of the Cu binding motif (C10S/C13S) were separated on native-PAGE either in the absence of the reducing agent DTT or in its presence. Indicated are the different oligomeric states of CopZ. (C) The Cu:protein ratio of purified CopZ was analyzed by atomic absorption spectroscopy. Wild type CopZ or the CopZ(C10S-C13S) mutant that lacked both conserved cysteine residues of the Cu binding motif, were directly analyzed (-Cu) or only after prior incubation with a 5-fold molar excess of Cu(I) and subsequent removal of unbound Cu(I) by gel filtration (+Cu). The molar Cu content was quantified by using a standard curve and the molar Cu:protein ratio was calculated. The values shown represent the mean of three independent experiments and the standard deviation is indicated by error bars.
    Figure Legend Snippet: CopZ is a Cu binding protein that forms redox-sensitive oligomers. (A) Purified CopZ was separated on native, non-reducing PAGE and after western transfer was decorated with α-CopZ antibodies (left panel). The dominant CopZ species are indicated by numbers (1–3); two additional minor species are indicated by (*), but those were not further analyzed. The dominant species 1–3 were gel-extracted from the native gel and separated on a second dimension SDS-PAGE under reducing conditions (right panel). The second dimension was stained with coomassie brilliant blue. Indicated are CopZ and a second band (?) that was not characterized further. (B) Purified CopZ and its derivatives lacking either one (C10S) or both conserved cysteines of the Cu binding motif (C10S/C13S) were separated on native-PAGE either in the absence of the reducing agent DTT or in its presence. Indicated are the different oligomeric states of CopZ. (C) The Cu:protein ratio of purified CopZ was analyzed by atomic absorption spectroscopy. Wild type CopZ or the CopZ(C10S-C13S) mutant that lacked both conserved cysteine residues of the Cu binding motif, were directly analyzed (-Cu) or only after prior incubation with a 5-fold molar excess of Cu(I) and subsequent removal of unbound Cu(I) by gel filtration (+Cu). The molar Cu content was quantified by using a standard curve and the molar Cu:protein ratio was calculated. The values shown represent the mean of three independent experiments and the standard deviation is indicated by error bars.

    Techniques Used: Binding Assay, Purification, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page, Staining, Clear Native PAGE, Atomic Absorption Spectroscopy, Mutagenesis, Incubation, Filtration, Standard Deviation

    CopZ forms a complex with the P 1B -type ATPase CcoI. (A) ICMs of the indicated strains were solubilized, and separated without further purification by BN-PAGE. After western transfer, potential CcoI and CopA complexes were visualized with antibodies against their C-terminal Myc-tags. (B) As in (A), but probing for CcoI complexes in membranes of the Δ copZ strain. (C) ICMs of the Δ ccoI strain complemented with p ccoI (upper two panels), the Δ ccoI strain (middle two panels) and of Δ copZ p ccoI (lower two panels) were solubilized and separated on BN-PAGE as in (A). The BN-PAGE gel lane was cut out, subsequently equilibrated in equilibration buffer containing SDS and urea and subjected to a 2 nd dimension SDS-PAGE. After western transfer, the membrane was horizontally cut and the upper part was decorated with α-Myc antibodies and the lower part with α-CopZ antibodies. Representative gels of at least three biological replicates are shown.
    Figure Legend Snippet: CopZ forms a complex with the P 1B -type ATPase CcoI. (A) ICMs of the indicated strains were solubilized, and separated without further purification by BN-PAGE. After western transfer, potential CcoI and CopA complexes were visualized with antibodies against their C-terminal Myc-tags. (B) As in (A), but probing for CcoI complexes in membranes of the Δ copZ strain. (C) ICMs of the Δ ccoI strain complemented with p ccoI (upper two panels), the Δ ccoI strain (middle two panels) and of Δ copZ p ccoI (lower two panels) were solubilized and separated on BN-PAGE as in (A). The BN-PAGE gel lane was cut out, subsequently equilibrated in equilibration buffer containing SDS and urea and subjected to a 2 nd dimension SDS-PAGE. After western transfer, the membrane was horizontally cut and the upper part was decorated with α-Myc antibodies and the lower part with α-CopZ antibodies. Representative gels of at least three biological replicates are shown.

    Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Western Blot, SDS Page

    31) Product Images from "Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability"

    Article Title: Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki169

    Serum starvation does not significantly alter the levels of PARN or translation initiation factors associated directly or indirectly with the cap. Hep G2 cells were grown in Eagle's MEM supplemented with 10% fetal calf serum (stimulated lanes) or cultured in serum-free media for 15 h (starved lanes). Total protein from S10 or S100 fractions was prepared and electrophoresed on polyacrylamide gels containing SDS. Western blotting was performed with the indicated antisera to detect PARN, eIF4E, eIF4G, PABP and 4E-BP1. Proteins were visualized with ECL reagents.
    Figure Legend Snippet: Serum starvation does not significantly alter the levels of PARN or translation initiation factors associated directly or indirectly with the cap. Hep G2 cells were grown in Eagle's MEM supplemented with 10% fetal calf serum (stimulated lanes) or cultured in serum-free media for 15 h (starved lanes). Total protein from S10 or S100 fractions was prepared and electrophoresed on polyacrylamide gels containing SDS. Western blotting was performed with the indicated antisera to detect PARN, eIF4E, eIF4G, PABP and 4E-BP1. Proteins were visualized with ECL reagents.

    Techniques Used: Cell Culture, Western Blot

    32) Product Images from "A pyro-phosphodegron controls MYC polyubiquitination to regulate cell survival"

    Article Title: A pyro-phosphodegron controls MYC polyubiquitination to regulate cell survival

    Journal: bioRxiv

    doi: 10.1101/2020.02.12.945634

    PEST domain pyrophosphorylation promotes F-box protein FBW7α binding and polyubiquitination of MYC. (A) Full-length mMYC-V5 protein and its mutant forms were transiently expressed in HEK293T cells. Numbers show mean fold change±SEM in expression level of individual mutants over the native protein (N=3). ( B, C ) Representative immunoblot for half-life measurement of mMYC-V5 forms. Cells were treated with 250 µg/mL CHX for the indicated time, and half-life was determined as described in Fig. 1C (N=3). ( D ) mMYC-V5 WT and its mutant forms were transiently expressed in HEK293T cells and treated as in Fig 1D , immunoprecipitated with anti-V5 antibody and immunoblotted with an anti-ubiquitin antibody (Ub-mMYC-V5) or anti-V5 antibody (N=3). ( E ) eGFP fused C-terminally with native or 3(S/T) mutant form of mMYC PEST domain (aa 202-268) was transiently expressed in HEK293T cells, immunoprecipitated, incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane, as described in Fig. 2A . Representative images show autoradiography to determine pyrophosphorylation (top) and immunoblotting with a GFP antibody (bottom) (N=2). ( F ) Representative immunoblots to assess the stability of eGFP, eGFP-mMYC PEST, or eGFP-mMYC PEST 3(S/T). HEK293T cells transiently expressing the different forms of eGFP were treated with 100 µg/mL cycloheximide (CHX) for the indicated time, lysed and immunoblotted to detect eGFP. GAPDH levels were monitored as a loading control (N=3). (G) Representative immunoblot showing the extent of phosphorylation at Thr58 and expression levels of endogenous MYC in Ip6k1 +/+ and Ip6k1 −/− MEFs. Numbers show mean fold change±SEM in Thr58 phosphorylation normalized to MYC levels in Ip6k1 −/− over Ip6k1 +/+ (N=3). (H) Representative immunoblots of interaction between mMYC and FBW7α. mMYC-V5 WT and pyrophosphorylation-deficient mutant mMYC-V5 3(S/T) were transiently co-expressed with c-Myc epitope-tagged FBW7α in HEK293T cells, immunoprecipitated with anti-V5 antibody, and probed to detect c-Myc epitope. The level of coimmunoprecipitated FBW7α was normalized to the level of immunoprecipitated MYC. Numbers show mean fold change±SEM in the extent of coimmunoprecipitation of FBW7α with 3(S/T) MYC compared with WT MYC (N=3) ( I ) Representative immunoblots of FBW7α interaction with eGFP fused with mMYC PEST domain. eGFP-mMYC PEST or eGFP-mMYC PEST 3(S/T) were transiently co-expressed with c-Myc epitope tagged FBW7α in HEK293T cells, immunoprecipitated with anti-GFP antibody, and probed to detect c-Myc epitope. No coimmunoprecipitation of FBW7α was detected with eGFP fused with pyrophosphorylation-deficient mMYC PEST 3(S/T) (N=3).
    Figure Legend Snippet: PEST domain pyrophosphorylation promotes F-box protein FBW7α binding and polyubiquitination of MYC. (A) Full-length mMYC-V5 protein and its mutant forms were transiently expressed in HEK293T cells. Numbers show mean fold change±SEM in expression level of individual mutants over the native protein (N=3). ( B, C ) Representative immunoblot for half-life measurement of mMYC-V5 forms. Cells were treated with 250 µg/mL CHX for the indicated time, and half-life was determined as described in Fig. 1C (N=3). ( D ) mMYC-V5 WT and its mutant forms were transiently expressed in HEK293T cells and treated as in Fig 1D , immunoprecipitated with anti-V5 antibody and immunoblotted with an anti-ubiquitin antibody (Ub-mMYC-V5) or anti-V5 antibody (N=3). ( E ) eGFP fused C-terminally with native or 3(S/T) mutant form of mMYC PEST domain (aa 202-268) was transiently expressed in HEK293T cells, immunoprecipitated, incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane, as described in Fig. 2A . Representative images show autoradiography to determine pyrophosphorylation (top) and immunoblotting with a GFP antibody (bottom) (N=2). ( F ) Representative immunoblots to assess the stability of eGFP, eGFP-mMYC PEST, or eGFP-mMYC PEST 3(S/T). HEK293T cells transiently expressing the different forms of eGFP were treated with 100 µg/mL cycloheximide (CHX) for the indicated time, lysed and immunoblotted to detect eGFP. GAPDH levels were monitored as a loading control (N=3). (G) Representative immunoblot showing the extent of phosphorylation at Thr58 and expression levels of endogenous MYC in Ip6k1 +/+ and Ip6k1 −/− MEFs. Numbers show mean fold change±SEM in Thr58 phosphorylation normalized to MYC levels in Ip6k1 −/− over Ip6k1 +/+ (N=3). (H) Representative immunoblots of interaction between mMYC and FBW7α. mMYC-V5 WT and pyrophosphorylation-deficient mutant mMYC-V5 3(S/T) were transiently co-expressed with c-Myc epitope-tagged FBW7α in HEK293T cells, immunoprecipitated with anti-V5 antibody, and probed to detect c-Myc epitope. The level of coimmunoprecipitated FBW7α was normalized to the level of immunoprecipitated MYC. Numbers show mean fold change±SEM in the extent of coimmunoprecipitation of FBW7α with 3(S/T) MYC compared with WT MYC (N=3) ( I ) Representative immunoblots of FBW7α interaction with eGFP fused with mMYC PEST domain. eGFP-mMYC PEST or eGFP-mMYC PEST 3(S/T) were transiently co-expressed with c-Myc epitope tagged FBW7α in HEK293T cells, immunoprecipitated with anti-GFP antibody, and probed to detect c-Myc epitope. No coimmunoprecipitation of FBW7α was detected with eGFP fused with pyrophosphorylation-deficient mMYC PEST 3(S/T) (N=3).

    Techniques Used: Binding Assay, Mutagenesis, Expressing, Immunoprecipitation, Incubation, Autoradiography, Western Blot

    MYC is pyrophosphorylated by 5-IP 7 in its central PEST domain. (A) Mouse MYC tagged C-terminally with V5 (mMYC-V5) was transiently expressed in HEK293T cells, immunoprecipitated, incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane (shown schematically on the left). Representative images show autoradiography to determine pyrophosphorylation (right) and immunoblotting with a V5-tag antibody (left) (N=3). Cells transfected with pCDNA3.1(+) plasmid (vector) served as a negative control. (B) Schematic on the left describes the back-pyrophosphorylation assay ( 23 ). Endogenous MYC from Ip6k1 +/+ and Ip6k1 −/− MEFs was immunoprecipitated and pyrophosphorylated in presence of 5[β- 32 P]IP 7 as in A . Representative images show autoradiography to detect pyrophosphorylation (right) and immunoblotting to detect MYC (left). Numbers show mean fold change±SEM in the extent of back-pyrophosphorylation in Ip6k1 −/− over Ip6k1 +/+ MEFs (N=3). (C) Domain map of MYC. Mouse MYC (mMYC) and human MYC (hMYC) show 91% identity in their PEST domains (protein-protein BLAST); all acidic Ser motifs (overlined) are conserved; pyrophosphorylated Ser cluster is in bold and underlined. (D) Purified, GST-tagged hMYC (GST-hMYC) PEST domain fragments were phosphorylated by CK2 in presence of unlabeled ATP, and then pyrophosphorylated by 5[β- 32 P]IP 7 (shown schematically on the left). Representative images show autoradiography to detect pyrophosphorylation (right) and immunoblotting with a GST antibody (left) (N=2). Line indicates removal of non-essential lanes from a single original gel. Start and end amino acid numbers for hMYC fragments are indicated in brackets. (E) Purified, GST-hMYC PEST domain (aa 201-268) corresponding to the native sequence, or with three Ser residues (249/250/252) mutated to Ala (3(S/A)) or Thr (3(S/T)), were treated as described in D (N=2). Proteins were detected by Ponceau S staining of the membrane. ( F ) Full-length mMYC-V5 protein (WT) or its mutant forms, Ser 249/250/252 replaced with Ala (3(S/A)), Asp (3(S/D)) or Thr (3(S/T)), were transiently expressed in HEK293T cells and treated as in A (N=2). Native mMYC shows pyrophosphorylation as in A , mMYC 3(S/T) shows faint pyrophosphorylation, whereas mMYC 3(S/A) and 3(S/D) are of the same intensity as non-specific bands (marked by an asterisk).
    Figure Legend Snippet: MYC is pyrophosphorylated by 5-IP 7 in its central PEST domain. (A) Mouse MYC tagged C-terminally with V5 (mMYC-V5) was transiently expressed in HEK293T cells, immunoprecipitated, incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane (shown schematically on the left). Representative images show autoradiography to determine pyrophosphorylation (right) and immunoblotting with a V5-tag antibody (left) (N=3). Cells transfected with pCDNA3.1(+) plasmid (vector) served as a negative control. (B) Schematic on the left describes the back-pyrophosphorylation assay ( 23 ). Endogenous MYC from Ip6k1 +/+ and Ip6k1 −/− MEFs was immunoprecipitated and pyrophosphorylated in presence of 5[β- 32 P]IP 7 as in A . Representative images show autoradiography to detect pyrophosphorylation (right) and immunoblotting to detect MYC (left). Numbers show mean fold change±SEM in the extent of back-pyrophosphorylation in Ip6k1 −/− over Ip6k1 +/+ MEFs (N=3). (C) Domain map of MYC. Mouse MYC (mMYC) and human MYC (hMYC) show 91% identity in their PEST domains (protein-protein BLAST); all acidic Ser motifs (overlined) are conserved; pyrophosphorylated Ser cluster is in bold and underlined. (D) Purified, GST-tagged hMYC (GST-hMYC) PEST domain fragments were phosphorylated by CK2 in presence of unlabeled ATP, and then pyrophosphorylated by 5[β- 32 P]IP 7 (shown schematically on the left). Representative images show autoradiography to detect pyrophosphorylation (right) and immunoblotting with a GST antibody (left) (N=2). Line indicates removal of non-essential lanes from a single original gel. Start and end amino acid numbers for hMYC fragments are indicated in brackets. (E) Purified, GST-hMYC PEST domain (aa 201-268) corresponding to the native sequence, or with three Ser residues (249/250/252) mutated to Ala (3(S/A)) or Thr (3(S/T)), were treated as described in D (N=2). Proteins were detected by Ponceau S staining of the membrane. ( F ) Full-length mMYC-V5 protein (WT) or its mutant forms, Ser 249/250/252 replaced with Ala (3(S/A)), Asp (3(S/D)) or Thr (3(S/T)), were transiently expressed in HEK293T cells and treated as in A (N=2). Native mMYC shows pyrophosphorylation as in A , mMYC 3(S/T) shows faint pyrophosphorylation, whereas mMYC 3(S/A) and 3(S/D) are of the same intensity as non-specific bands (marked by an asterisk).

    Techniques Used: Immunoprecipitation, Incubation, Autoradiography, Transfection, Plasmid Preparation, Negative Control, Purification, Sequencing, Staining, Mutagenesis

    33) Product Images from "Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex"

    Article Title: Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr705

    Phosphorylation of P20. ( A ) In vitro phosphorylation. Purified rP20 was co-incubated in protein extracts of BaMV and satBaMV co-infected N. benthamiana leaves in the absence or presence of 100 mM EDTA and [γ- 32 P]ATP or [γ- 32 P]GTP and analyzed by 12.5% SDS–PAGE and autoradiography. Reaction mixture lacking rP20 or leaf protein extract served as controls. ( C ) In vivo phosphorylation. N. benthamiana protoplasts were mock-inoculated, inoculated with BaMV viral RNA alone or co-inoculated with BaMV RNA and satBaMV RNA transcripts by electroporation and cultured with [ 33 P]orthophosphate. After 20 h inoculation, protoplasts were harvested, lysed, total proteins were immunoprecipitated with anti-P20 serum, analyzed by 12.5% SDS–PAGE and detected by autoradiography. ( B and D ), Immunodetection of P20 phosphorylated in vitro and in vivo with anti-P20 serum.
    Figure Legend Snippet: Phosphorylation of P20. ( A ) In vitro phosphorylation. Purified rP20 was co-incubated in protein extracts of BaMV and satBaMV co-infected N. benthamiana leaves in the absence or presence of 100 mM EDTA and [γ- 32 P]ATP or [γ- 32 P]GTP and analyzed by 12.5% SDS–PAGE and autoradiography. Reaction mixture lacking rP20 or leaf protein extract served as controls. ( C ) In vivo phosphorylation. N. benthamiana protoplasts were mock-inoculated, inoculated with BaMV viral RNA alone or co-inoculated with BaMV RNA and satBaMV RNA transcripts by electroporation and cultured with [ 33 P]orthophosphate. After 20 h inoculation, protoplasts were harvested, lysed, total proteins were immunoprecipitated with anti-P20 serum, analyzed by 12.5% SDS–PAGE and detected by autoradiography. ( B and D ), Immunodetection of P20 phosphorylated in vitro and in vivo with anti-P20 serum.

    Techniques Used: In Vitro, Purification, Incubation, Infection, SDS Page, Autoradiography, In Vivo, Electroporation, Cell Culture, Immunoprecipitation, Immunodetection

    34) Product Images from "cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolor A3(2)"

    Article Title: cmdABCDEF, a cluster of genes encoding membrane proteins for differentiation and antibiotic production in Streptomyces coelicolor A3(2)

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-157

    Localization of CmdB protein, characterization of its functional domain, and detection of cmdB transcription . (A) Localization of CmdB protein. Cell lysates of strain M145 and that were treated with 0.5 M KCl or 5 mM EDTA-Na, were centrifuged to obtain supernatants (S) and pellets (P) for Western blotting with CmdB polyclonal antibody. Total cell lysates was a positive control. (B) Mutations of conserved residues in domains of the CmdB protein blocked its function. Plasmid pFX101 derivatives containing the site-mutated cmdB genes were introduced by conjugation into the cmdB null mutant. Strains were grown on MS at 30°C for 3 days. (C) RT-PCR to detect transcription of cmdB . Total RNA was isolated from MS medium grown for 16, 26, 40, 50, 62 and 74 h, and reverse-transcribed into cDNAs for PCR amplification. Transcription of 16S rRNA gene was used as an internal control.
    Figure Legend Snippet: Localization of CmdB protein, characterization of its functional domain, and detection of cmdB transcription . (A) Localization of CmdB protein. Cell lysates of strain M145 and that were treated with 0.5 M KCl or 5 mM EDTA-Na, were centrifuged to obtain supernatants (S) and pellets (P) for Western blotting with CmdB polyclonal antibody. Total cell lysates was a positive control. (B) Mutations of conserved residues in domains of the CmdB protein blocked its function. Plasmid pFX101 derivatives containing the site-mutated cmdB genes were introduced by conjugation into the cmdB null mutant. Strains were grown on MS at 30°C for 3 days. (C) RT-PCR to detect transcription of cmdB . Total RNA was isolated from MS medium grown for 16, 26, 40, 50, 62 and 74 h, and reverse-transcribed into cDNAs for PCR amplification. Transcription of 16S rRNA gene was used as an internal control.

    Techniques Used: Functional Assay, Western Blot, Positive Control, Plasmid Preparation, Conjugation Assay, Mutagenesis, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification

    35) Product Images from "The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists"

    Article Title: The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20170097

    IRAK1 activation does not require its phosphorylation or ubiquitylation. ( A ) IRAK1 KO IL-1R cells were transfected with 5 µg of DNA of control empty vector (EV) or HA-IRAK1, and IRAK1 then immunoprecipitated from 0.5 mg of cell extract protein. The IPs were incubated with PP1γ (10 U) in the presence or absence of microcystin (10 µM) and denatured in SDS. Following SDS–PAGE and transfer to PVDF membranes, immunoblotting was performed with anti-IRAK1. ( B ) As in A , except that, after PP1γ treatment, the immunoprecipitates were washed, incubated for 1 h with 10 µM microcystin, and IRAK1 was assayed with GST-Pellino1 and Mg[γ 32 P-ATP] as substrates in the absence (−) or presence (+) of JNK-IN-7 (1 µM) and in the presence (+) of IRAK4-IN-1 (1 µM) to inactivate co-immunoprecipitating IRAK4. The presence of IRAK1 in the immunoprecipitates was also analyzed by immunoblotting. ( C ) IL-1R cells were stimulated with IL-1β and IRAK1 immunoprecipitated from 1 mg of cell extract protein and incubated with PP1γ (10 U) and USP2 (1.15 µg). The immunoprecipitates were washed, incubated for 1 h with 1 µM microcystin to inactivate any residual PP1γ, and IRAK1 assayed with GST-Pellino1 and Mg[γ 32 P-ATP] as substrates in the presence (+) of IRAK4-IN-1 (1 µM) to inactivate any co-immunoprecipitating IRAK4. ( D ) The autoradiogram from C and one other independent experiment were scanned and IRAK1 activity quantitated after incubation with or without USP2 and PP1γ. The results are presented as a % of that measured without USP2 and PP1γ treatment.
    Figure Legend Snippet: IRAK1 activation does not require its phosphorylation or ubiquitylation. ( A ) IRAK1 KO IL-1R cells were transfected with 5 µg of DNA of control empty vector (EV) or HA-IRAK1, and IRAK1 then immunoprecipitated from 0.5 mg of cell extract protein. The IPs were incubated with PP1γ (10 U) in the presence or absence of microcystin (10 µM) and denatured in SDS. Following SDS–PAGE and transfer to PVDF membranes, immunoblotting was performed with anti-IRAK1. ( B ) As in A , except that, after PP1γ treatment, the immunoprecipitates were washed, incubated for 1 h with 10 µM microcystin, and IRAK1 was assayed with GST-Pellino1 and Mg[γ 32 P-ATP] as substrates in the absence (−) or presence (+) of JNK-IN-7 (1 µM) and in the presence (+) of IRAK4-IN-1 (1 µM) to inactivate co-immunoprecipitating IRAK4. The presence of IRAK1 in the immunoprecipitates was also analyzed by immunoblotting. ( C ) IL-1R cells were stimulated with IL-1β and IRAK1 immunoprecipitated from 1 mg of cell extract protein and incubated with PP1γ (10 U) and USP2 (1.15 µg). The immunoprecipitates were washed, incubated for 1 h with 1 µM microcystin to inactivate any residual PP1γ, and IRAK1 assayed with GST-Pellino1 and Mg[γ 32 P-ATP] as substrates in the presence (+) of IRAK4-IN-1 (1 µM) to inactivate any co-immunoprecipitating IRAK4. ( D ) The autoradiogram from C and one other independent experiment were scanned and IRAK1 activity quantitated after incubation with or without USP2 and PP1γ. The results are presented as a % of that measured without USP2 and PP1γ treatment.

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, SDS Page, Activity Assay

    36) Product Images from "LccA, an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii ▿ ▿ †"

    Article Title: LccA, an Archaeal Laccase Secreted as a Highly Stable Glycoprotein into the Extracellular Medium by Haloferax volcanii ▿ ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01757-09

    LccA purified to electrophoretic homogeneity from the culture broth of H. volcanii US02. Shown is reducing 10% SDS-PAGE of LccA at various stages of purification, including ethanol precipitate (EP) (lane 1) and MonoQ 10/10, pH 8.4 (MQ) (lane 2), as indicated. Molecular mass standards are on the left. Proteins were stained with Coomassie brilliant blue R-250.
    Figure Legend Snippet: LccA purified to electrophoretic homogeneity from the culture broth of H. volcanii US02. Shown is reducing 10% SDS-PAGE of LccA at various stages of purification, including ethanol precipitate (EP) (lane 1) and MonoQ 10/10, pH 8.4 (MQ) (lane 2), as indicated. Molecular mass standards are on the left. Proteins were stained with Coomassie brilliant blue R-250.

    Techniques Used: Purification, SDS Page, Staining

    LccA purified from US02 is a glycoprotein. (A) Native gels of LccA at its final stages of purification from US02 (Superdex 200 HR 10/30, lanes 1 and 2) and SB01 (MonoQ 10/10, pH 8.4, lanes 3 and 4). The gels were stained for total protein with Coomassie brilliant blue R-250 (CB) and for laccase activity by in-gel oxidation of SGZ (SGZ), as indicated. Prestained kaleidoscope and low-range protein standards (Bio-Rad) were included for US02 and SB01, respectively, as indicated at the left of each gel. (B) Pro-Q Emerald glycoprotein analysis of US02-purified LccA. Proteins were separated by reducing 10% SDS-PAGE. The gels were stained for glycosylation with Pro-Q Emerald (left) and for total protein with Sypro-Ruby (right), as indicated. The proteins included carbonic anhydrase (CA) (lanes 1); T. versicolor laccase (TvLc) (lanes 2); LccA MonoQ 10/10, pH 8.4, fractions (HvLc MQ ) (lanes 3); LccA ethanol precipitate (HvLc EP ) (lanes 4); and CandyCane molecular mass standards (Molecular Probes), including a mixture of glyosylated (G) and nonglycosylated proteins, as indicated (lanes 5). (C) Deglycosylation of LccA. LccA purified from US02 was treated with TFMS on ice for 0 h (lanes 1), 10 h (lanes 2), and 3 h (lane 3) and separated by reducing 6 and 10% SDS-PAGE, as indicated (see Materials and Methods for details).
    Figure Legend Snippet: LccA purified from US02 is a glycoprotein. (A) Native gels of LccA at its final stages of purification from US02 (Superdex 200 HR 10/30, lanes 1 and 2) and SB01 (MonoQ 10/10, pH 8.4, lanes 3 and 4). The gels were stained for total protein with Coomassie brilliant blue R-250 (CB) and for laccase activity by in-gel oxidation of SGZ (SGZ), as indicated. Prestained kaleidoscope and low-range protein standards (Bio-Rad) were included for US02 and SB01, respectively, as indicated at the left of each gel. (B) Pro-Q Emerald glycoprotein analysis of US02-purified LccA. Proteins were separated by reducing 10% SDS-PAGE. The gels were stained for glycosylation with Pro-Q Emerald (left) and for total protein with Sypro-Ruby (right), as indicated. The proteins included carbonic anhydrase (CA) (lanes 1); T. versicolor laccase (TvLc) (lanes 2); LccA MonoQ 10/10, pH 8.4, fractions (HvLc MQ ) (lanes 3); LccA ethanol precipitate (HvLc EP ) (lanes 4); and CandyCane molecular mass standards (Molecular Probes), including a mixture of glyosylated (G) and nonglycosylated proteins, as indicated (lanes 5). (C) Deglycosylation of LccA. LccA purified from US02 was treated with TFMS on ice for 0 h (lanes 1), 10 h (lanes 2), and 3 h (lane 3) and separated by reducing 6 and 10% SDS-PAGE, as indicated (see Materials and Methods for details).

    Techniques Used: Purification, Staining, Activity Assay, SDS Page

    37) Product Images from "A Comprehensive Approach to Identification of Surface-Exposed, Outer Membrane-Spanning Proteins of Leptospira interrogans"

    Article Title: A Comprehensive Approach to Identification of Surface-Exposed, Outer Membrane-Spanning Proteins of Leptospira interrogans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006071

    Surface localization of L. interrogans serovar Copenhageni strain Fiocruz L1–130 proteins by proteinase K treatment. Whole intact spirochetes were incubated with different concentrations of proteinase K, equivalents of 1×10 8 of leptospires per lane separated by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), transferred to a PVDF membrane, and probed with polyclonal rabbit antisera against: (A) OmpL37; (B) OmpL47; (C) OmpL54; (D) OmpL36; (E) FlaA1 and LipL31; (F) LipL46. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.
    Figure Legend Snippet: Surface localization of L. interrogans serovar Copenhageni strain Fiocruz L1–130 proteins by proteinase K treatment. Whole intact spirochetes were incubated with different concentrations of proteinase K, equivalents of 1×10 8 of leptospires per lane separated by gel electrophoresis (Bis-Tris 4–12% NuPage gel, Novex), transferred to a PVDF membrane, and probed with polyclonal rabbit antisera against: (A) OmpL37; (B) OmpL47; (C) OmpL54; (D) OmpL36; (E) FlaA1 and LipL31; (F) LipL46. The identities of individual proteins are indicated on the right, and the positions of molecular mass standard (in kilodaltons) are indicated on the left.

    Techniques Used: Incubation, Nucleic Acid Electrophoresis

    38) Product Images from "Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport"

    Article Title: Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20160610

    Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by CK2, and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.
    Figure Legend Snippet: Inositol pyrophosphates pyrophosphorylate dynein IC. ( A ) Bacterially expressed and purified IC(1–111) was phosphorylated in vitro by CK2, and phosphosite identification was contracted out to the Taplin Mass Spectrometry Facility, Harvard Medical School. The MS/MS spectrum is shown for the doubly phosphorylated peptide corresponding to residues 42–55 of mouse IC-2C (EAAVpSVQEEpSDLEK). The sequence shows the peptide fragmentation pattern, and the table shows masses of all b and y ions, highlighting the ions obtained in the spectrum. Arrows indicate fragment ions containing phosphorylated Ser residues. The mass of fragment y5 indicates phosphorylation of Ser51, and the masses of y10 and b10 correspond to phosphorylation of Ser46 and Ser51. ( B ) Bacterially expressed and purified GST or GST-tagged IC(1–70), IC(1–111), and IC(1–111)S51A were prephosphorylated with CK2 and unlabeled ATP and incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and the proteins were detected by Ponceau S staining (left). The phosphorimager scan was subjected to ‘levels’ adjustment in Adobe Photoshop to improve visualization. The image intensity of the pyrophosphorylated protein was normalized to the corresponding total protein. The pyrophosphorylation intensity of each IC fragment was compared with GST. Data are mean ± range from two independent experiments. ( C ) Back-pyrophosphorylation of endogenous dynein IC by IP 7 . Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with 5[β- 32 P]IP 7 , resolved by NuPAGE, and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right), and proteins were detected by western blotting (left). The image intensity of the pyrophosphorylated protein was normalized to the corresponding immunoprecipitated protein. The fold change in the extent of pyrophosphorylation of IC in Ip6k1 −/− compared with Ip6k1 +/+ MEFs is indicated. Data are mean ± SEM from three independent experiments. ( D ) Back-phosphorylation of endogenous dynein IC by CK2. Dynein IC immunoprecipitated from Ip6k1 +/+ and Ip6k1 −/− MEFs was incubated with CK2 and [γ- 32 P]ATP. Proteins were resolved and detected as in ( C ). The fold change in phosphorylation was calculated as in ( C ). Data are mean ± range from two independent experiments.

    Techniques Used: Purification, In Vitro, Mass Spectrometry, Sequencing, Incubation, Staining, Immunoprecipitation, Western Blot

    39) Product Images from "Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae"

    Article Title: Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae

    Journal: Biochemical Journal

    doi: 10.1042/BJ20140798

    IP 7 pyrophosphorylates RNA Pol I subunits ( A ) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). ( B ) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in ( A ). ( C ) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). ( D–F ) Pyrophosphorylation, as in ( A ), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. ( G ) Pyrophosphorylation, as in ( A ), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in ( A ), ( F ) and ( G ) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels ( A ) and ( F ) indicate the removal of non-essential lanes from a single original gel.
    Figure Legend Snippet: IP 7 pyrophosphorylates RNA Pol I subunits ( A ) Purified, GST-tagged, full-length (FL) proteins Uaf30, A34.5, and A43, and the indicated fragments of A135 and A190, were incubated with 5[β- 32 P]IP 7 . Proteins were resolved using NuPAGE and transferred to a PVDF membrane. Pyrophosphorylation was detected by phosphorimager scanning (right) and proteins were detected by Western blotting (left). ( B ) Purified, GST-tagged, A190 fragments were pyrophosphorylated as in ( A ). ( C ) Purified GST-tagged A190 fragments corresponding to the native sequence and the indicated serine-to-alanine point mutants were pyrophosphorylated as in (A). ( D–F ) Pyrophosphorylation, as in ( A ), of purified, GST-tagged, FL fragments, and the indicated serine-to-alanine point mutants of A43. ( G ) Pyrophosphorylation, as in ( A ), of purified GST-tagged FL in-frame deletion, a C-terminally truncated fragment and the indicated serine-to-alanine point mutants of A34.5. To improve visualization, phosphorimager scans in ( A ), ( F ) and ( G ) were subjected to tonal range adjustment of the whole image using Adobe Photoshop (level adjustment). The start and end amino acid numbers of protein fragments are indicated in brackets. The dividing lines between lanes in panels ( A ) and ( F ) indicate the removal of non-essential lanes from a single original gel.

    Techniques Used: Purification, Incubation, Western Blot, Sequencing

    40) Product Images from "Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells"

    Article Title: Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015227

    Secretion of sdAbs to the extracellular medium with T3SS of EPEC. ( A ) Schematic representation of the T3SS-complex encoded by EPEC, labeling the essential ATPase EscN, the extracellular EspA filament and the secreted EspB and EspD translocators. The secretion of T3sNbs from the cytoplasm of the bacteria to the extracellular medium is indicated. ( B ) Coomassie staining of TCA-precipitated proteins found in the extracellular media of cultures of wt EPEC or Δ escN strains carrying plasmids pSA10, pT3sVamy, or pT3sVgfp, as indicated. Cultures were grown at 37°C in DMEM and induced with 0.1 mM IPTG for 4 h. The protein bands of EspA, EspB, EspD, and that of the Sec-dependent autotransporter EspC, are labeled. Size in kDa of protein standards for SDS-PAGE are shown on the right. ( C ) Western blot analysis of the proteins found in extracellular media (Culture supernantants; lanes 1–6) and cells (bacterial lysates; lanes 7–12) from the same cultures as in (B). WB developed with mAbs anti-E-tag (top panels) or anti-GroEL (bottom panels) to control the absence of cytoplasmic proteins in the extracellular media. ( D ) Binding activity of the secreted sdAbs. ELISA with His-tag purified T3sVgfp (left) and T3sVamy (right), at the indicated concentrations (nM), against their cognate antigens (GFP or Amy) and BSA (negative control). Bound T3sV HH s developed with anti-E-tag mAb-POD and their Optical Density (O.D.) determined at 490 nm.
    Figure Legend Snippet: Secretion of sdAbs to the extracellular medium with T3SS of EPEC. ( A ) Schematic representation of the T3SS-complex encoded by EPEC, labeling the essential ATPase EscN, the extracellular EspA filament and the secreted EspB and EspD translocators. The secretion of T3sNbs from the cytoplasm of the bacteria to the extracellular medium is indicated. ( B ) Coomassie staining of TCA-precipitated proteins found in the extracellular media of cultures of wt EPEC or Δ escN strains carrying plasmids pSA10, pT3sVamy, or pT3sVgfp, as indicated. Cultures were grown at 37°C in DMEM and induced with 0.1 mM IPTG for 4 h. The protein bands of EspA, EspB, EspD, and that of the Sec-dependent autotransporter EspC, are labeled. Size in kDa of protein standards for SDS-PAGE are shown on the right. ( C ) Western blot analysis of the proteins found in extracellular media (Culture supernantants; lanes 1–6) and cells (bacterial lysates; lanes 7–12) from the same cultures as in (B). WB developed with mAbs anti-E-tag (top panels) or anti-GroEL (bottom panels) to control the absence of cytoplasmic proteins in the extracellular media. ( D ) Binding activity of the secreted sdAbs. ELISA with His-tag purified T3sVgfp (left) and T3sVamy (right), at the indicated concentrations (nM), against their cognate antigens (GFP or Amy) and BSA (negative control). Bound T3sV HH s developed with anti-E-tag mAb-POD and their Optical Density (O.D.) determined at 490 nm.

    Techniques Used: Labeling, Staining, Size-exclusion Chromatography, SDS Page, Western Blot, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification, Negative Control

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    Western Blot:

    Article Title: Nucleolar Reorganization Upon Site-Specific Double-Strand Break Induction
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    Incubation:

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    SDS Page:

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    GE Healthcare pvdf membranes
    Time course of Klotho-mediated phosphorylation of endogenous 14-3-3ζ (Ser-58) effect on 14-3-3ζ monomer levels in HEK 293 cells. (A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the <t>Trx/ASK1</t> complex protection against oxidative stress occurred ( Fig 1 ). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 k Da protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto <t>PVDF</t> membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in S3 Table .
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    Time course of Klotho-mediated phosphorylation of endogenous 14-3-3ζ (Ser-58) effect on 14-3-3ζ monomer levels in HEK 293 cells. (A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the Trx/ASK1 complex protection against oxidative stress occurred ( Fig 1 ). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 k Da protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in S3 Table .

    Journal: PLoS ONE

    Article Title: Klotho Regulates 14-3-3ζ Monomerization and Binding to the ASK1 Signaling Complex in Response to Oxidative Stress

    doi: 10.1371/journal.pone.0141968

    Figure Lengend Snippet: Time course of Klotho-mediated phosphorylation of endogenous 14-3-3ζ (Ser-58) effect on 14-3-3ζ monomer levels in HEK 293 cells. (A) Plot describing time-dependent 14-3-3ζ phosphorylation mediated by Klotho was demonstrated by adding 200 pM of the recombinant protein to the cultured cells at the indicated times. Peak phosphorylation times (i.e. 30–45 min) are within the range where the Trx/ASK1 complex protection against oxidative stress occurred ( Fig 1 ). Data are reported as ± SEM. (B) Plot of 14-3-3ζ monomer level in HEK 293 cells treated with either secreted Klotho or buffer control for 40 min. Shown beside plot is a native Western blot of the samples as described in Materials and Methods. The 14-3-3ζ antibody recognizes a predominant ~30 k Da protein band representing the expected size of the monomer. Replicate samples were separated under SDS-PAGE, electroblotted onto PVDF membrane and probed with same antibody to account for lysate levels of total 14-3-3ζ. Digitized values of the WB of monomer levels are shown in S3 Table .

    Article Snippet: Resolved proteins were Western transferred onto PVDF membranes and probed with Trx, ASK1, or 14-3-3ζ antibodies as described above.

    Techniques: Recombinant, Cell Culture, Western Blot, SDS Page

    Specificity of the anti-TEKT2BP1 antibody. (A) Recombinant GST-fused proteins were produced in E. coli and separated by SDS-PAGE. Separated proteins were stained with Coomassie brilliant blue (upper panel) or transferred to a PVDF membrane for immunoblotting

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Molecular Cloning and Subcellular Localization of Tektin2-Binding Protein 1 (Ccdc 172) in Rat Spermatozoa

    doi: 10.1369/0022155413520607

    Figure Lengend Snippet: Specificity of the anti-TEKT2BP1 antibody. (A) Recombinant GST-fused proteins were produced in E. coli and separated by SDS-PAGE. Separated proteins were stained with Coomassie brilliant blue (upper panel) or transferred to a PVDF membrane for immunoblotting

    Article Snippet: Separated proteins were either stained with Coomassie brilliant blue or transferred to PVDF membranes (Hybond-P, GE Health).

    Techniques: Recombinant, Produced, SDS Page, Staining

    Gel filtration analysis of the interaction between Photofrin and recombinant procaspase-3-D 3 A. Recombinant procaspase-3-D 3 A alone (1 mg), Photofrin alone (18.13 μ g) or recombinant procaspase-3-D 3 A (1 mg) plus Photofrin (18.13 μ g) (molar ratio 1 : 1) were applied to Superose 12 columns for gel filtration analysis, as described. ( a ) Each fraction was subjected to optical density measurement (280 nm) for procaspase-3-D 3 A proteins and fluorescence measurement (excitation 400 nm and emission 630 nm) for Photofrin. ( b ) Fractions were also directly spotted onto a PVDF membrane, and procaspase-3-D 3 A and Photofrin signals were detected by immunoblotting and fluorescence scanning (Typhoon 9400), respectively. ( c ) Procaspase-3-D 3 A, bovine serum albumin (BSA), ovalbumin, casein (10 μ g of each protein) or Photofrin (5 μ g) were spotted onto a PVDF membrane. The membrane was incubated with Photofrin-containing solution (100 μ g in 10 ml distilled water) at room temperature in the dark for 3 h, washed thrice for 10 min with distilled water and thrice for 10 min with TTBS buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, and 0.05% Tween 20). The washed membrane was air-dried and then scanned by a Typhoon 9400 fluorescence scanner

    Journal: Cell Death & Disease

    Article Title: Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

    doi: 10.1038/cddis.2012.85

    Figure Lengend Snippet: Gel filtration analysis of the interaction between Photofrin and recombinant procaspase-3-D 3 A. Recombinant procaspase-3-D 3 A alone (1 mg), Photofrin alone (18.13 μ g) or recombinant procaspase-3-D 3 A (1 mg) plus Photofrin (18.13 μ g) (molar ratio 1 : 1) were applied to Superose 12 columns for gel filtration analysis, as described. ( a ) Each fraction was subjected to optical density measurement (280 nm) for procaspase-3-D 3 A proteins and fluorescence measurement (excitation 400 nm and emission 630 nm) for Photofrin. ( b ) Fractions were also directly spotted onto a PVDF membrane, and procaspase-3-D 3 A and Photofrin signals were detected by immunoblotting and fluorescence scanning (Typhoon 9400), respectively. ( c ) Procaspase-3-D 3 A, bovine serum albumin (BSA), ovalbumin, casein (10 μ g of each protein) or Photofrin (5 μ g) were spotted onto a PVDF membrane. The membrane was incubated with Photofrin-containing solution (100 μ g in 10 ml distilled water) at room temperature in the dark for 3 h, washed thrice for 10 min with distilled water and thrice for 10 min with TTBS buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, and 0.05% Tween 20). The washed membrane was air-dried and then scanned by a Typhoon 9400 fluorescence scanner

    Article Snippet: Fractions were also directly spotted onto PVDF membranes, and procaspase-3-D3 A and Photofrin signals were detected by immunoblotting and fluorescence scanning (Typhoon 9400; GE Healthcare), respectively.

    Techniques: Filtration, Recombinant, Fluorescence, Incubation

    Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on PVDF membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments

    Journal: Cell Death & Disease

    Article Title: Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

    doi: 10.1038/cddis.2010.1

    Figure Lengend Snippet: Galectin-1 (Gal-1)-induced phosphorylation of c-Jun N-terminal kinase 1 (JNK1) and JNK2 ( a ) and JNK activation with c-Jun(1-169)-GST ( b ), and c-Jun(1-89)-GST ( c ) as kinase substrates. Jurkat E6.1 cells (2 × 10 6 per ml RPMI 1640 medium) were incubated with protein kinase C-θ (PKCθ) inhibitor and PKCδ inhibitor rottlerin for 1 h, with the sphingomyelinase inhibitors desipramine and imipramine for 2 h, as well as with the ATP-competitive inhibitor for JNK SP600125 and for mitogen-activated protein kinase kinase 4 (MKK4) myricetin for 30 min as indicated. Control cells were incubated in medium alone. Cells were then stimulated with gal-1 without and in the presence of lactose or asialofetuin as indicated in panels a , b , and c . ( a ) For immunoblot analysis cell extract proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Blots were analyzed with a phospho-JNK (Thr183/Tyr185) monoclonal antibody (mAb). The bands were luminographically visualized on X-ray films using ECL Plus reagents. Equal loading of gel lanes was verified by reprobing the blots for expression of β -actin. ( b ) After termination of the kinase reactions with 3 × SDS sample buffer, samples were electrophoretically separated and blotted on PVDF membranes. The [ 32 P]-labeled substrate c-Jun(1-169)-GST was recorded by autoradiography. To control loading, we separated 50 μ g cell extract protein/lane and blotted it on PVDF membranes. Membranes were probed with a JNK1 polyclonal antibody (pAb). ( c ) After termination of the kinase reactions, samples were separated and blotted on Hybond ECL membranes. Blots were analyzed for substrate phosphorylation with a phospho-c-Jun (Ser63) pAb. The bands were luminographically visualized on X-ray films using ECL Plus reagents. Shown are representative blots from three independent experiments

    Article Snippet: Fetal calf serum (FCS), kanamycin, RPMI 1640 medium were from Gibco BRL (Eggenstein, Germany), enhanced chemiluminescence (ECL) detection reagents, Hybond ECL nitrocellulose membranes, protein G agarose, PVDF membranes, and [γ -32 P]ATP were from GE Healthcare Europe (Freiburg, Germany).

    Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Expressing, Labeling, Autoradiography