streptavidin sepharose beads  (GE Healthcare)

 
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    Streptavidin Sepharose High Performance
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    Streptavidin Sepharose High Performance affinity resin
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    Structured Review

    GE Healthcare streptavidin sepharose beads
    ENaC ubiquitylation in whole cell lysates and at the cell surface. A , Hek293 cells were transiently transfected with either wild-type or KR mutant ENaC channels. The ubiquitylated and the total amount of ENaC expressed in Hek293 cells were visualized by Western blotting of immunoprecipitated α-(HA), β-(c-Myc), and γ-(VSV)-ENaC, with either anti ubiquitin or anti ENaC antibodies. B , cells were biotinylated, lysed and then immunoprecipitated with HA tag (for α), c-Myc tag (for β), and VSV tag (for γ). The channels present at the cells surface were recovered using <t>streptavidin-Sepharose</t> beads. Biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. ENaC KR : K to R mutation on all cytoplasmic lysines. fu : mutation on furin sites. The nature of a fragment seen occasionally at ∼70 kDa in the ENaC blots is not known (*). C , Hek293 cells were transiently transfected with ENaC WT +/− Nedd4-2. Cells were biotinylated 24 h after transfection. Then they were lysed and an immunoprecipitation was performed with anti-HA to recover αENaC. The cell surface channels were recovered using streptavidin beads. Then proteins were run on SDS/PAGE and Western blot were performed as describe. Quantification of three individual experiments of the ratio ubiquitylated/full-length αENaC was calculated. They were normalized to αENaC full-length and displayed as mean ± S.E. ( n = 3 experiments, *, p

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    1) Product Images from "Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change *"

    Article Title: Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.176156

    ENaC ubiquitylation in whole cell lysates and at the cell surface. A , Hek293 cells were transiently transfected with either wild-type or KR mutant ENaC channels. The ubiquitylated and the total amount of ENaC expressed in Hek293 cells were visualized by Western blotting of immunoprecipitated α-(HA), β-(c-Myc), and γ-(VSV)-ENaC, with either anti ubiquitin or anti ENaC antibodies. B , cells were biotinylated, lysed and then immunoprecipitated with HA tag (for α), c-Myc tag (for β), and VSV tag (for γ). The channels present at the cells surface were recovered using streptavidin-Sepharose beads. Biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. ENaC KR : K to R mutation on all cytoplasmic lysines. fu : mutation on furin sites. The nature of a fragment seen occasionally at ∼70 kDa in the ENaC blots is not known (*). C , Hek293 cells were transiently transfected with ENaC WT +/− Nedd4-2. Cells were biotinylated 24 h after transfection. Then they were lysed and an immunoprecipitation was performed with anti-HA to recover αENaC. The cell surface channels were recovered using streptavidin beads. Then proteins were run on SDS/PAGE and Western blot were performed as describe. Quantification of three individual experiments of the ratio ubiquitylated/full-length αENaC was calculated. They were normalized to αENaC full-length and displayed as mean ± S.E. ( n = 3 experiments, *, p
    Figure Legend Snippet: ENaC ubiquitylation in whole cell lysates and at the cell surface. A , Hek293 cells were transiently transfected with either wild-type or KR mutant ENaC channels. The ubiquitylated and the total amount of ENaC expressed in Hek293 cells were visualized by Western blotting of immunoprecipitated α-(HA), β-(c-Myc), and γ-(VSV)-ENaC, with either anti ubiquitin or anti ENaC antibodies. B , cells were biotinylated, lysed and then immunoprecipitated with HA tag (for α), c-Myc tag (for β), and VSV tag (for γ). The channels present at the cells surface were recovered using streptavidin-Sepharose beads. Biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. ENaC KR : K to R mutation on all cytoplasmic lysines. fu : mutation on furin sites. The nature of a fragment seen occasionally at ∼70 kDa in the ENaC blots is not known (*). C , Hek293 cells were transiently transfected with ENaC WT +/− Nedd4-2. Cells were biotinylated 24 h after transfection. Then they were lysed and an immunoprecipitation was performed with anti-HA to recover αENaC. The cell surface channels were recovered using streptavidin beads. Then proteins were run on SDS/PAGE and Western blot were performed as describe. Quantification of three individual experiments of the ratio ubiquitylated/full-length αENaC was calculated. They were normalized to αENaC full-length and displayed as mean ± S.E. ( n = 3 experiments, *, p

    Techniques Used: Transfection, Mutagenesis, Western Blot, Immunoprecipitation, SDS Page

    Proteolytic cleavage of wild-type and mutant channels mutated on cytoplasmic lysines. A , Hek293 cells were transiently transfected with either wild-type ( W ) or cytoplasmic lysine mutant ( K ) ENaC. 24 h after transfection, cells were biotinylated, recovered with streptavidin-Sepharose and analyzed by SDS-PAGE/Western blotting using anti α-, or γ-ENaC antibodies as indicated. fl : full-length α- or γ-ENaC; arrow : cleaved α- or γ-ENaC. α- and γENaC antibodies crossreact with endogenous proteins, as shown in lane 1 . In our previous work we have provided evidence that these do not represent endogenous ENaC. B , quantification of the ratio of cleaved to full-length αENaC (as described under “Experimental Procedures”), normalized to wild-type ENaC (condition 2), and displayed as mean ± S.E. ( n = 3 experiments; *, p
    Figure Legend Snippet: Proteolytic cleavage of wild-type and mutant channels mutated on cytoplasmic lysines. A , Hek293 cells were transiently transfected with either wild-type ( W ) or cytoplasmic lysine mutant ( K ) ENaC. 24 h after transfection, cells were biotinylated, recovered with streptavidin-Sepharose and analyzed by SDS-PAGE/Western blotting using anti α-, or γ-ENaC antibodies as indicated. fl : full-length α- or γ-ENaC; arrow : cleaved α- or γ-ENaC. α- and γENaC antibodies crossreact with endogenous proteins, as shown in lane 1 . In our previous work we have provided evidence that these do not represent endogenous ENaC. B , quantification of the ratio of cleaved to full-length αENaC (as described under “Experimental Procedures”), normalized to wild-type ENaC (condition 2), and displayed as mean ± S.E. ( n = 3 experiments; *, p

    Techniques Used: Mutagenesis, Transfection, SDS Page, Western Blot

    2) Product Images from "The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿"

    Article Title: The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00567-10

    Direct interaction of the VirB promoter with BvrR. (A) Purified BvrR protein or whole-bacterium lysates of B. abortus 2308 were incubated with streptavidin-Sepharose beads preloaded with a biotin-labeled amplicon comprising the virB promoter region or
    Figure Legend Snippet: Direct interaction of the VirB promoter with BvrR. (A) Purified BvrR protein or whole-bacterium lysates of B. abortus 2308 were incubated with streptavidin-Sepharose beads preloaded with a biotin-labeled amplicon comprising the virB promoter region or

    Techniques Used: Purification, Incubation, Labeling, Amplification

    3) Product Images from "Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania"

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007237

    Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.
    Figure Legend Snippet: Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.

    Techniques Used: Transgenic Assay, Expressing, Fractionation, SDS Page, Migration, Western Blot, Flow Cytometry

    The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.
    Figure Legend Snippet: The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.

    Techniques Used: Mutagenesis, Migration, SDS Page, Transgenic Assay, Expressing, Western Blot, Copurification, Flow Cytometry, Confocal Microscopy, Incubation, Labeling, Staining

    Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p
    Figure Legend Snippet: Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Luciferase, Software

    4) Product Images from "Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide"

    Article Title: Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0709-4

    Nutrient starvation upregulates nSMase2. PC12 cells were starved with HBSS for the indicated times. a Induction of autophagy by nutrient starvation in PC12 cells. Degradation of p62 and LC3 turnover were detected by immunoblotting for assessing autophagic flux. For LC3 turnover assays, cells were starved with HBSS with or without 50 μM CQ for the indicated times. b Activation of nSMase2 by starvation. Specific activity of nSMase2 was analyzed using [ 14 C]-labeled sphingomyelin. c Increase in nSMase2 expression by starvation. Protein expression levels of nSMase2 in starved cells were determined by immunoblotting and were normalized to β-actin levels. d No changes in nSMase2 mRNA levels were induced by starvation. The mRNA levels of nSMase2 were measured by quantitative real-time PCR and were normalized to Hprt1 . e Starvation-induced phosphorylation of nSMase2 at a serine residue. PC12 cells were starved with HBSS for the indicated time, and cell lysates were incubated with biotin-conjugated sphingomyelin (the nSMase2 substrate) followed by pull-down with streptavidin-sepharose beads. The pellets were analyzed using immunoblots to detect serine phosphorylation of nSMase2. The data are presented as the mean ± SEM of three independent experiments. Significant differences, * p
    Figure Legend Snippet: Nutrient starvation upregulates nSMase2. PC12 cells were starved with HBSS for the indicated times. a Induction of autophagy by nutrient starvation in PC12 cells. Degradation of p62 and LC3 turnover were detected by immunoblotting for assessing autophagic flux. For LC3 turnover assays, cells were starved with HBSS with or without 50 μM CQ for the indicated times. b Activation of nSMase2 by starvation. Specific activity of nSMase2 was analyzed using [ 14 C]-labeled sphingomyelin. c Increase in nSMase2 expression by starvation. Protein expression levels of nSMase2 in starved cells were determined by immunoblotting and were normalized to β-actin levels. d No changes in nSMase2 mRNA levels were induced by starvation. The mRNA levels of nSMase2 were measured by quantitative real-time PCR and were normalized to Hprt1 . e Starvation-induced phosphorylation of nSMase2 at a serine residue. PC12 cells were starved with HBSS for the indicated time, and cell lysates were incubated with biotin-conjugated sphingomyelin (the nSMase2 substrate) followed by pull-down with streptavidin-sepharose beads. The pellets were analyzed using immunoblots to detect serine phosphorylation of nSMase2. The data are presented as the mean ± SEM of three independent experiments. Significant differences, * p

    Techniques Used: Activation Assay, Activity Assay, Labeling, Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot

    5) Product Images from "Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization"

    Article Title: Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization

    Journal: Journal of Virology

    doi: 10.1128/JVI.00574-17

    Processing and functional characteristics of surface-expressed GP1 N-glycosylation sites. (A) Vero cells were transfected with the indicated FLAG-tagged LASV GP variant or the negative control. After 36 h, cells were subjected to surface biotinylation. Surface-expressed biotinylated proteins were concentrated using streptavidin Sepharose beads. Precipitated proteins were separated by SDS-PAGE. Immunoblot assays were carried out to detect LASV GP surface-expressed protein using an anti-FLAG antibody, M2. The immunoblot shown is representative of four trials. (B) Microphotographs of Vero cells cotransfected with plasmid DNA encoding LASV GP construct and GFP. Cell-to-cell fusion was assessed 3 h following low-pH-medium shock; magnification, ×20. Representative fields of view are shown. (C) Fusion data for each construct was quantified by counting unfused cells and comparing the numbers to those in mock-transfected wells. Quantified fusion data for each construct were normalized to LASV wt-GPC-3xFLAG. Cleavage efficiency was normalized to FLAG-tagged GP using densitometry analysis. (D) Parental GP transduction efficiency in HAP1 and HAP1-ΔDAG1 cells. VSVΔG-GFP pseudoparticles containing LASV GP were added to both cells, and the GFP-positive cells were enumerated in a flow cytometer. The percentage of the cell population that was GFP positive is shown. (E) VSVΔG-GFP pseudoparticles containing LASV GP or N-glycosylation mutants were used to transduce HAP1 and HAP1-ΔDAG1 cells. The GFP-positive cells were enumerated in a flow cytometer. Transduction efficiencies were normalized to parental LASV GP particle transduction values in each respective cell type. All data are based on the averages and standard errors of the means from at least three replicate experiments.
    Figure Legend Snippet: Processing and functional characteristics of surface-expressed GP1 N-glycosylation sites. (A) Vero cells were transfected with the indicated FLAG-tagged LASV GP variant or the negative control. After 36 h, cells were subjected to surface biotinylation. Surface-expressed biotinylated proteins were concentrated using streptavidin Sepharose beads. Precipitated proteins were separated by SDS-PAGE. Immunoblot assays were carried out to detect LASV GP surface-expressed protein using an anti-FLAG antibody, M2. The immunoblot shown is representative of four trials. (B) Microphotographs of Vero cells cotransfected with plasmid DNA encoding LASV GP construct and GFP. Cell-to-cell fusion was assessed 3 h following low-pH-medium shock; magnification, ×20. Representative fields of view are shown. (C) Fusion data for each construct was quantified by counting unfused cells and comparing the numbers to those in mock-transfected wells. Quantified fusion data for each construct were normalized to LASV wt-GPC-3xFLAG. Cleavage efficiency was normalized to FLAG-tagged GP using densitometry analysis. (D) Parental GP transduction efficiency in HAP1 and HAP1-ΔDAG1 cells. VSVΔG-GFP pseudoparticles containing LASV GP were added to both cells, and the GFP-positive cells were enumerated in a flow cytometer. The percentage of the cell population that was GFP positive is shown. (E) VSVΔG-GFP pseudoparticles containing LASV GP or N-glycosylation mutants were used to transduce HAP1 and HAP1-ΔDAG1 cells. The GFP-positive cells were enumerated in a flow cytometer. Transduction efficiencies were normalized to parental LASV GP particle transduction values in each respective cell type. All data are based on the averages and standard errors of the means from at least three replicate experiments.

    Techniques Used: Functional Assay, Transfection, Variant Assay, Negative Control, SDS Page, Plasmid Preparation, Construct, Gel Permeation Chromatography, Transduction, Flow Cytometry, Cytometry

    6) Product Images from "Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs"

    Article Title: Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

    Journal: Nature Communications

    doi: 10.1038/ncomms3980

    HnRNPA2B1 specifically binds EXOmiRNAs. Exosome extracts were incubated with streptavidin beads coated with either a biotinylated EXOmiRNA (miR-198), a biotinylated CLmiRNA (miR-17) or a negative control (poly-A), and with non-coated beads (beads). The graphs represent the extracted ion chromatogram traces of the monoisotopic peaks corresponding to the indicated peptides, identified from hnRNPA2B1or from a non-specifically binding protein (nucleolin).
    Figure Legend Snippet: HnRNPA2B1 specifically binds EXOmiRNAs. Exosome extracts were incubated with streptavidin beads coated with either a biotinylated EXOmiRNA (miR-198), a biotinylated CLmiRNA (miR-17) or a negative control (poly-A), and with non-coated beads (beads). The graphs represent the extracted ion chromatogram traces of the monoisotopic peaks corresponding to the indicated peptides, identified from hnRNPA2B1or from a non-specifically binding protein (nucleolin).

    Techniques Used: Incubation, Negative Control, Binding Assay

    7) Product Images from "KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression"

    Article Title: KLHL22 maintains PD-1 homeostasis and prevents excessive T cell suppression

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

    doi: 10.1073/pnas.2004570117

    KLHL22 is a major PD-1–associated protein. ( A . ( B . ( C and D ) Venn diagram showing the overlap of two MS results. Proteins appearing in the MS results of both the Jurkat PD-1–FLAG cells and HEK293T PD-1–SFB cells. The results from the overlap shown in C are presented in a 2D graph in D . ( E ) 6-Kelch repeats in KLHL22 are required for the interaction between KLHL22 and PD-1. HEK293T cells were cotransfected with untagged PD-1 (PD-1 without an artificial tag) and PD-L1-SFB, SFB-KLHL22, or SFB-KLHL22Δ6K. The cell lysates were subjected to pull-down assays with S protein Sepharose and immunoblotted with the indicated antibodies. ( F ) HEK293T cells were cotransfected with PD-1–SFB and Myc-KLHL22, Myc-KLHL22Δ6K, Myc-KLHL9, or Myc-KLHL13. The cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. ( G ) Lysates of Jurkat cells stably expressing PD-1–FLAG were immunoprecipitated with FLAG-M2 beads or protein G beads with IgG and subjected to immunoblotting with the indicated antibodies. Jurkat cells were stimulated by PMA (50 ng/mL) and ionomycin (1 μM) for 12 h. ( H ) The endogenous interaction of PD-1 and KLHL22 in healthy human PBMCs using KLHL22 antibody pull-down. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) for 12 h, 24 h, or 36 h. ( I ) Endogenous PD-1 associates with endogenous KLHL22 in healthy human PBMCs. Human healthy PBMCs lysates were immunoprecipitated with an anti–PD-1 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) or anti-CD3 (1 μg/mL)/anti-CD28 (2 μg/mL) for 24 h. The third group was also treated with PD-1 antibody (2 μg/mL) for 24 h. ( J ) Endogenous KLHL22 associates with endogenous PD-1 in healthy human PBMCs. CD28 and CTLA4 were tested simultaneously and showed negative results. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (0.5 μg/mL or 1 μg/mL) and anti-CD28 (2 μg/mL) for 24 h.
    Figure Legend Snippet: KLHL22 is a major PD-1–associated protein. ( A . ( B . ( C and D ) Venn diagram showing the overlap of two MS results. Proteins appearing in the MS results of both the Jurkat PD-1–FLAG cells and HEK293T PD-1–SFB cells. The results from the overlap shown in C are presented in a 2D graph in D . ( E ) 6-Kelch repeats in KLHL22 are required for the interaction between KLHL22 and PD-1. HEK293T cells were cotransfected with untagged PD-1 (PD-1 without an artificial tag) and PD-L1-SFB, SFB-KLHL22, or SFB-KLHL22Δ6K. The cell lysates were subjected to pull-down assays with S protein Sepharose and immunoblotted with the indicated antibodies. ( F ) HEK293T cells were cotransfected with PD-1–SFB and Myc-KLHL22, Myc-KLHL22Δ6K, Myc-KLHL9, or Myc-KLHL13. The cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. ( G ) Lysates of Jurkat cells stably expressing PD-1–FLAG were immunoprecipitated with FLAG-M2 beads or protein G beads with IgG and subjected to immunoblotting with the indicated antibodies. Jurkat cells were stimulated by PMA (50 ng/mL) and ionomycin (1 μM) for 12 h. ( H ) The endogenous interaction of PD-1 and KLHL22 in healthy human PBMCs using KLHL22 antibody pull-down. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) for 12 h, 24 h, or 36 h. ( I ) Endogenous PD-1 associates with endogenous KLHL22 in healthy human PBMCs. Human healthy PBMCs lysates were immunoprecipitated with an anti–PD-1 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (1 μg/mL) or anti-CD3 (1 μg/mL)/anti-CD28 (2 μg/mL) for 24 h. The third group was also treated with PD-1 antibody (2 μg/mL) for 24 h. ( J ) Endogenous KLHL22 associates with endogenous PD-1 in healthy human PBMCs. CD28 and CTLA4 were tested simultaneously and showed negative results. Human healthy PBMCs lysates were immunoprecipitated with an anti-KLHL22 antibody or IgG and subjected to immunoblotting with the indicated antibodies. PBMCs were stimulated by anti-CD3 (0.5 μg/mL or 1 μg/mL) and anti-CD28 (2 μg/mL) for 24 h.

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation

    KLHL22 mediates polyubiquitination-directed degradation of incompletely glycosylated PD-1. ( A ) PD-1 ubiquitination is inhibited by KLHL22 depletion. Control or KLHL22 -specific siRNA was transfected into HEK293T cells stably expressing PD-1–SFB in the presence of MG132 (1 µM 24 h). Cell lysates were subjected to pull-down assays by S-protein Sepharose and immunoblotted with the indicated antibodies. ( B ) PD-1 ubiquitination is inhibited upon MLN4924 treatment. HEK293T cells stably expressing PD-1–SFB were treated with BFA (1 µM), MLN4924 (1 µM), and MG132 (1 µM) as indicated for 12 h, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with anti-FLAG and antiubiquitin antibodies. ( C ) Incompletely glycosylated PD-1 is unstable in vivo. PD-1–SFB stable cells were incubated in medium containing 10 μg/mL cycloheximide (CHX) in the presence or absence of BFA (1 µM) for the indicated time. Western blotting was carried out using the indicated antibodies. ( D ) The KLHL22/CUL3/RBX1 complex ubiquitinates PD-1 in vivo. CUL3, RBX1, and either KLHL22 or KLHL22Δ6K were overexpressed in HEK293T cells stably expressing PD-1–SFB, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. ( E ) Only 48K ubiquitin can be conjugated to PD-1. For ubiquitination mutants transfected into HEK293T cells stably expressing PD-1–SFB, all lysine’s were mutated to arginine except Lys48 (48K) or Lys63 (63K). Cells were treated with MG132 (1 µM, 24 h). Ubiquitination of PD-1 was detected by immunoblotting with antiubiquitin antibody. Cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with antiubiquitin and anti-FLAG antibodies. ( F ) The ubiquitination of PD-1 on K210R and K233R is significantly reduced. PD-1–SFB (WT), PD-1–SFB (K210R), or PD-1–SFB (K233R) was transfected into HEK293T cells treated with BFA (1 µM), MLN4924 (1 µM) and MG132 (1 µM) as indicated for 12 h. The resulting cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies.
    Figure Legend Snippet: KLHL22 mediates polyubiquitination-directed degradation of incompletely glycosylated PD-1. ( A ) PD-1 ubiquitination is inhibited by KLHL22 depletion. Control or KLHL22 -specific siRNA was transfected into HEK293T cells stably expressing PD-1–SFB in the presence of MG132 (1 µM 24 h). Cell lysates were subjected to pull-down assays by S-protein Sepharose and immunoblotted with the indicated antibodies. ( B ) PD-1 ubiquitination is inhibited upon MLN4924 treatment. HEK293T cells stably expressing PD-1–SFB were treated with BFA (1 µM), MLN4924 (1 µM), and MG132 (1 µM) as indicated for 12 h, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with anti-FLAG and antiubiquitin antibodies. ( C ) Incompletely glycosylated PD-1 is unstable in vivo. PD-1–SFB stable cells were incubated in medium containing 10 μg/mL cycloheximide (CHX) in the presence or absence of BFA (1 µM) for the indicated time. Western blotting was carried out using the indicated antibodies. ( D ) The KLHL22/CUL3/RBX1 complex ubiquitinates PD-1 in vivo. CUL3, RBX1, and either KLHL22 or KLHL22Δ6K were overexpressed in HEK293T cells stably expressing PD-1–SFB, and cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies. ( E ) Only 48K ubiquitin can be conjugated to PD-1. For ubiquitination mutants transfected into HEK293T cells stably expressing PD-1–SFB, all lysine’s were mutated to arginine except Lys48 (48K) or Lys63 (63K). Cells were treated with MG132 (1 µM, 24 h). Ubiquitination of PD-1 was detected by immunoblotting with antiubiquitin antibody. Cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with antiubiquitin and anti-FLAG antibodies. ( F ) The ubiquitination of PD-1 on K210R and K233R is significantly reduced. PD-1–SFB (WT), PD-1–SFB (K210R), or PD-1–SFB (K233R) was transfected into HEK293T cells treated with BFA (1 µM), MLN4924 (1 µM) and MG132 (1 µM) as indicated for 12 h. The resulting cell lysates were subjected to pull-down assays with S-protein Sepharose and immunoblotted with the indicated antibodies.

    Techniques Used: Transfection, Stable Transfection, Expressing, In Vivo, Incubation, Western Blot

    8) Product Images from "A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways"

    Article Title: A novel 4E-interacting protein in Leishmania is involved in stage-specific translation pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr555

    Proteins associated with LeishIF4E-4 in promastigotes. ( A ) Pull-down experiments with the SBP-tagged LeishIF4E-4 from L. amazonensis promastigotes was done using affinity purification over streptavidin–Sepharose beads. Aliquots of the soluble extract (S, 1%), the flow-through (F, 1%), the final wash (W, 20%) and eluted proteins (E, 20%) were separated by SDS–PAGE (10–15%) and subjected to western blot analysis using specific antibodies against LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1. Densitometric analysis that was normalized to the total protein amounts showed that the elution fraction contained 16, 9 and 3.4% of the total LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1, respectively. The arrows indicate the specific reaction of the antibodies. ( B ) The yeast two-hybrid assay was performed by cotransfecting wild-type YRG-2 yeast strain with pBD fused to LeishIF4E-4, LeishIF4E-4 W163A mutant, LeishIF4A-1, LeishPABP-1, LeishIF4G-3 or yPAB1 and pAD-LeishIF4G-3, pAD-LeishIF4E-4 or pAD-LeishIF4E-4Δ1-86. Cells were cultured under restrictive (−His) and non-restrictive (+His) growth conditions, and spotted as described in ‘Materials and Methods’ section. Positive and negative controls were supplied by the manufacturer and expression of the tested proteins was verified by western blot analysis ( Supplementary Figure S7 ).
    Figure Legend Snippet: Proteins associated with LeishIF4E-4 in promastigotes. ( A ) Pull-down experiments with the SBP-tagged LeishIF4E-4 from L. amazonensis promastigotes was done using affinity purification over streptavidin–Sepharose beads. Aliquots of the soluble extract (S, 1%), the flow-through (F, 1%), the final wash (W, 20%) and eluted proteins (E, 20%) were separated by SDS–PAGE (10–15%) and subjected to western blot analysis using specific antibodies against LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1. Densitometric analysis that was normalized to the total protein amounts showed that the elution fraction contained 16, 9 and 3.4% of the total LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1, respectively. The arrows indicate the specific reaction of the antibodies. ( B ) The yeast two-hybrid assay was performed by cotransfecting wild-type YRG-2 yeast strain with pBD fused to LeishIF4E-4, LeishIF4E-4 W163A mutant, LeishIF4A-1, LeishPABP-1, LeishIF4G-3 or yPAB1 and pAD-LeishIF4G-3, pAD-LeishIF4E-4 or pAD-LeishIF4E-4Δ1-86. Cells were cultured under restrictive (−His) and non-restrictive (+His) growth conditions, and spotted as described in ‘Materials and Methods’ section. Positive and negative controls were supplied by the manufacturer and expression of the tested proteins was verified by western blot analysis ( Supplementary Figure S7 ).

    Techniques Used: Affinity Purification, Flow Cytometry, SDS Page, Western Blot, Y2H Assay, Mutagenesis, Cell Culture, Expressing

    Characterization of the LeishIF4F complex during stage differentiation. ( A ) Expression levels of LeishIF4F subunits in wild-type L. amazonensis , exposed or not to heat shock and differentiated in vitro . Cells were lysed and samples with 50 µg protein from the supernatants were resolved on SDS–PAGE (10–15%) and subjected to western blot analysis using specific antibodies against GP46, HSP100, LeishIF4E-1 to -4, LeishIF4G-3 and LeishIF4A-1. P, promastigotes from late log phase cultures; HS, late log promastigotes following a transient exposure to 33°C for 2 h; A, axenic amastigotes 9 days after differentiation. Coomassie staining of Tubulin was used to verify the protein loads. ( B ) Pull-down experiments with TAP tagged LeishIF4E-1 or LeishIF4E-4 from L. major promastigotes, exposed or not to heat shock (35°C, 2 h). Cell extracts were affinity purified over streptavidin-Sepharose beads and then over m 7 GTP-Sepharose column. The pulled-down proteins were analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1. ( C ) m 7 GTP pulldown assays with wild-type L. amazonensis promastigotes, promastigotes after 2 h at 33°C and axenic-amastigotes, 9 days after differentiation. The cells were lyzed and loaded on a m 7 GTP-Sepharose column. The eluted proteins were analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4E-4 and LeishIF4G-3. ( D ) Pull-down experiments in axenic-amastigotes of L. amazonensis expressing SBP tagged LeishIF4E-1, 9 days after differentiation. Proteins were purified and analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4G-3 and LeishIF4A-1.
    Figure Legend Snippet: Characterization of the LeishIF4F complex during stage differentiation. ( A ) Expression levels of LeishIF4F subunits in wild-type L. amazonensis , exposed or not to heat shock and differentiated in vitro . Cells were lysed and samples with 50 µg protein from the supernatants were resolved on SDS–PAGE (10–15%) and subjected to western blot analysis using specific antibodies against GP46, HSP100, LeishIF4E-1 to -4, LeishIF4G-3 and LeishIF4A-1. P, promastigotes from late log phase cultures; HS, late log promastigotes following a transient exposure to 33°C for 2 h; A, axenic amastigotes 9 days after differentiation. Coomassie staining of Tubulin was used to verify the protein loads. ( B ) Pull-down experiments with TAP tagged LeishIF4E-1 or LeishIF4E-4 from L. major promastigotes, exposed or not to heat shock (35°C, 2 h). Cell extracts were affinity purified over streptavidin-Sepharose beads and then over m 7 GTP-Sepharose column. The pulled-down proteins were analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4E-4, LeishIF4G-3 and LeishIF4A-1. ( C ) m 7 GTP pulldown assays with wild-type L. amazonensis promastigotes, promastigotes after 2 h at 33°C and axenic-amastigotes, 9 days after differentiation. The cells were lyzed and loaded on a m 7 GTP-Sepharose column. The eluted proteins were analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4E-4 and LeishIF4G-3. ( D ) Pull-down experiments in axenic-amastigotes of L. amazonensis expressing SBP tagged LeishIF4E-1, 9 days after differentiation. Proteins were purified and analyzed as described in Figure 1 A, using specific antibodies against LeishIF4E-1, LeishIF4G-3 and LeishIF4A-1.

    Techniques Used: Expressing, In Vitro, SDS Page, Western Blot, Staining, Affinity Purification, Purification

    9) Product Images from "Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities"

    Article Title: Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-017-0204-z

    HAS1-synthesized HA interacts with RHAMM. a RHAMM and its splice variants are associated with cellular HA (synthesized from HAS1 overexpression) during mitosis and G1/S phase. HeLa cells were co-transfected with plasmids expressing A2-tagged HAS1 (A2-HAS1) and full-length GFP-tagged RHAMM (RHAMM-GFP) or the splice-variants of RHAMM (RHAMM-Ex4-GFP). Cell populations were synchronized in Mitosis or G1/S using thymidine block and synchronization was verified using flow cytometry (Supplementary Fig. 3B). Total cellular HA was isolated using biotinylated bovine HA-binding-protein and streptavidin-conjugated magnetic beads. The isolated beads were treated (+) with hyaluronidase (HAase). Samples were subjected to immunoblotting for RHAMM and HAS1. b BRCA1 interacted with RHAMM isoforms but not with the other HA-binding protein Neurocan. GFP-tagged RHAMM isoforms and GFP-ΔNeurocan were expressed in HeLa cells and HA-binding proteins were isolated from the cell lysates using biotinylated-HA as “bait”. Immunoblotting of pull-down material with and without HAase treatment revealed that endogenous BRCA1 were found to be associated with RHAMM isoforms (but not with Neurocan), suggesting that BRCA1 may interact directly with RHAMM
    Figure Legend Snippet: HAS1-synthesized HA interacts with RHAMM. a RHAMM and its splice variants are associated with cellular HA (synthesized from HAS1 overexpression) during mitosis and G1/S phase. HeLa cells were co-transfected with plasmids expressing A2-tagged HAS1 (A2-HAS1) and full-length GFP-tagged RHAMM (RHAMM-GFP) or the splice-variants of RHAMM (RHAMM-Ex4-GFP). Cell populations were synchronized in Mitosis or G1/S using thymidine block and synchronization was verified using flow cytometry (Supplementary Fig. 3B). Total cellular HA was isolated using biotinylated bovine HA-binding-protein and streptavidin-conjugated magnetic beads. The isolated beads were treated (+) with hyaluronidase (HAase). Samples were subjected to immunoblotting for RHAMM and HAS1. b BRCA1 interacted with RHAMM isoforms but not with the other HA-binding protein Neurocan. GFP-tagged RHAMM isoforms and GFP-ΔNeurocan were expressed in HeLa cells and HA-binding proteins were isolated from the cell lysates using biotinylated-HA as “bait”. Immunoblotting of pull-down material with and without HAase treatment revealed that endogenous BRCA1 were found to be associated with RHAMM isoforms (but not with Neurocan), suggesting that BRCA1 may interact directly with RHAMM

    Techniques Used: Synthesized, Over Expression, Transfection, Expressing, Blocking Assay, Flow Cytometry, Isolation, Binding Assay, Magnetic Beads

    10) Product Images from "Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs"

    Article Title: Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

    Journal: Nature Communications

    doi: 10.1038/ncomms3980

    HnRNPA2B1 specifically binds EXOmiRNAs. Exosome extracts were incubated with streptavidin beads coated with either a biotinylated EXOmiRNA (miR-198), a biotinylated CLmiRNA (miR-17) or a negative control (poly-A), and with non-coated beads (beads). The graphs represent the extracted ion chromatogram traces of the monoisotopic peaks corresponding to the indicated peptides, identified from hnRNPA2B1or from a non-specifically binding protein (nucleolin).
    Figure Legend Snippet: HnRNPA2B1 specifically binds EXOmiRNAs. Exosome extracts were incubated with streptavidin beads coated with either a biotinylated EXOmiRNA (miR-198), a biotinylated CLmiRNA (miR-17) or a negative control (poly-A), and with non-coated beads (beads). The graphs represent the extracted ion chromatogram traces of the monoisotopic peaks corresponding to the indicated peptides, identified from hnRNPA2B1or from a non-specifically binding protein (nucleolin).

    Techniques Used: Incubation, Negative Control, Binding Assay

    11) Product Images from "Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization"

    Article Title: Mutational Analysis of Lassa Virus Glycoprotein Highlights Regions Required for Alpha-Dystroglycan Utilization

    Journal: Journal of Virology

    doi: 10.1128/JVI.00574-17

    Processing and functional characteristics of surface-expressed GP1 N-glycosylation sites. (A) Vero cells were transfected with the indicated FLAG-tagged LASV GP variant or the negative control. After 36 h, cells were subjected to surface biotinylation. Surface-expressed biotinylated proteins were concentrated using streptavidin Sepharose beads. Precipitated proteins were separated by SDS-PAGE. Immunoblot assays were carried out to detect LASV GP surface-expressed protein using an anti-FLAG antibody, M2. The immunoblot shown is representative of four trials. (B) Microphotographs of Vero cells cotransfected with plasmid DNA encoding LASV GP construct and GFP. Cell-to-cell fusion was assessed 3 h following low-pH-medium shock; magnification, ×20. Representative fields of view are shown. (C) Fusion data for each construct was quantified by counting unfused cells and comparing the numbers to those in mock-transfected wells. Quantified fusion data for each construct were normalized to LASV wt-GPC-3xFLAG. Cleavage efficiency was normalized to FLAG-tagged GP using densitometry analysis. (D) Parental GP transduction efficiency in HAP1 and HAP1-ΔDAG1 cells. VSVΔG-GFP pseudoparticles containing LASV GP were added to both cells, and the GFP-positive cells were enumerated in a flow cytometer. The percentage of the cell population that was GFP positive is shown. (E) VSVΔG-GFP pseudoparticles containing LASV GP or N-glycosylation mutants were used to transduce HAP1 and HAP1-ΔDAG1 cells. The GFP-positive cells were enumerated in a flow cytometer. Transduction efficiencies were normalized to parental LASV GP particle transduction values in each respective cell type. All data are based on the averages and standard errors of the means from at least three replicate experiments.
    Figure Legend Snippet: Processing and functional characteristics of surface-expressed GP1 N-glycosylation sites. (A) Vero cells were transfected with the indicated FLAG-tagged LASV GP variant or the negative control. After 36 h, cells were subjected to surface biotinylation. Surface-expressed biotinylated proteins were concentrated using streptavidin Sepharose beads. Precipitated proteins were separated by SDS-PAGE. Immunoblot assays were carried out to detect LASV GP surface-expressed protein using an anti-FLAG antibody, M2. The immunoblot shown is representative of four trials. (B) Microphotographs of Vero cells cotransfected with plasmid DNA encoding LASV GP construct and GFP. Cell-to-cell fusion was assessed 3 h following low-pH-medium shock; magnification, ×20. Representative fields of view are shown. (C) Fusion data for each construct was quantified by counting unfused cells and comparing the numbers to those in mock-transfected wells. Quantified fusion data for each construct were normalized to LASV wt-GPC-3xFLAG. Cleavage efficiency was normalized to FLAG-tagged GP using densitometry analysis. (D) Parental GP transduction efficiency in HAP1 and HAP1-ΔDAG1 cells. VSVΔG-GFP pseudoparticles containing LASV GP were added to both cells, and the GFP-positive cells were enumerated in a flow cytometer. The percentage of the cell population that was GFP positive is shown. (E) VSVΔG-GFP pseudoparticles containing LASV GP or N-glycosylation mutants were used to transduce HAP1 and HAP1-ΔDAG1 cells. The GFP-positive cells were enumerated in a flow cytometer. Transduction efficiencies were normalized to parental LASV GP particle transduction values in each respective cell type. All data are based on the averages and standard errors of the means from at least three replicate experiments.

    Techniques Used: Functional Assay, Transfection, Variant Assay, Negative Control, SDS Page, Plasmid Preparation, Construct, Gel Permeation Chromatography, Transduction, Flow Cytometry, Cytometry

    12) Product Images from "RAB31 marks and controls an ESCRT-independent exosome pathway"

    Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

    Journal: Cell Research

    doi: 10.1038/s41422-020-00409-1

    RAB31 recruits TBC1D2B to inactivate RAB7 suppressing the fusion of MVEs with lysosomes. a Immunofluorescence of endogenous RAB7 (red) and endogenous CD63 (magenta) with GFP-RAB31 WT (green) in the indicated stable NCI-H1975 cells. b Western blotting analyses of whole-cell lysates (WCL) and streptavidin pull-down (PD) from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. c Immunofluorescence of endogenous RAB7 (red) and HA-RILP (magenta) with GFP-RAB31 (green) in the indicated stable HeLa cells transiently expressing HA-RILP. d Western blotting analyses of WCL and streptavidin PD from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. e Immunofluorescence of endogenous RAB7 (red) and endogenous TBC1D2B (magenta) with GFP-RAB31 (green) in the indicated stable NCI-H1975 cells. f Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB31 (green) in NCI-H1975 cells. g Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. h Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB7 (green) in NCI-H1975 cells stably expressing shNC or shRAB31. i Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. j Western blotting analyses of WCL and GTP agarose PD at their endogenous levels from NCI-H1975 and MDA-MB231 cells stably expressing shNC or shRAB31. Scale bars, 10 μm.
    Figure Legend Snippet: RAB31 recruits TBC1D2B to inactivate RAB7 suppressing the fusion of MVEs with lysosomes. a Immunofluorescence of endogenous RAB7 (red) and endogenous CD63 (magenta) with GFP-RAB31 WT (green) in the indicated stable NCI-H1975 cells. b Western blotting analyses of whole-cell lysates (WCL) and streptavidin pull-down (PD) from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. c Immunofluorescence of endogenous RAB7 (red) and HA-RILP (magenta) with GFP-RAB31 (green) in the indicated stable HeLa cells transiently expressing HA-RILP. d Western blotting analyses of WCL and streptavidin PD from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. e Immunofluorescence of endogenous RAB7 (red) and endogenous TBC1D2B (magenta) with GFP-RAB31 (green) in the indicated stable NCI-H1975 cells. f Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB31 (green) in NCI-H1975 cells. g Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. h Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB7 (green) in NCI-H1975 cells stably expressing shNC or shRAB31. i Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. j Western blotting analyses of WCL and GTP agarose PD at their endogenous levels from NCI-H1975 and MDA-MB231 cells stably expressing shNC or shRAB31. Scale bars, 10 μm.

    Techniques Used: Immunofluorescence, Western Blot, Expressing, Stable Transfection, Multiple Displacement Amplification

    13) Product Images from "ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1"

    Article Title: ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1

    Journal: Nature cell biology

    doi: 10.1038/ncb3013

    ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1 ( a ) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10 μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates were immunoprecipitated with the CHK1 antibody and immunoblotted with antibodies indicated. ( b ) A partial list of ZEB1-associated proteins. ( c, d ) 293T cells were transfected with SFB-ZEB1 ( c ) or SFB-USP7 ( d ), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies indicated. ( e ) Top: bacterially purified GST-USP7 was incubated with amylose resin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteins retained on the amylose resin were immunoblotted with the USP7 antibody. Bottom: bacterially purified recombinant proteins were analyzed by SDS-PAGE and Coomassie blue staining. * indicates the predicted position. ( f ) 293T cells were transfected with SFB-USP7 and treated with cycloheximide (CHX). Cells were harvested at different time points and immunoblotted with antibodies indicated. ( g, h ) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7, g ) or transduced with ZEB1 shRNA (sh-ZEB1, h ), and treated with cycloheximide. Cells were harvested at different time points and immunoblotted with antibodies indicated. ( i ) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into 293T cells. Lysates from cells with or without 6 Gy IR treatment were immunoprecipitated with the CHK1 antibody and immunoblotted with the HA antibody. Cells were treated with MG132 (10 μM) for 6 hr before harvest. ( j ) Top: ubiquitinated CHK1 was incubated with SFB-GFP control or SFB-USP7 purified with streptavidin-sepharose beads from 293T cells with or without ZEB1 co-transfection. The reaction mixture was then immunoprecipitated with the FLAG antibody and immunoblotted with the CHK1 antibody. Bottom: purified SFB-USP7 was immunoblotted with antibodies to ZEB1 and USP7. ( k ) Clonogenic survival assays of USP7 siRNA-transfected SUM159-P2 cells. n = 3 wells per group. Data in k are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1 ( a ) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10 μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates were immunoprecipitated with the CHK1 antibody and immunoblotted with antibodies indicated. ( b ) A partial list of ZEB1-associated proteins. ( c, d ) 293T cells were transfected with SFB-ZEB1 ( c ) or SFB-USP7 ( d ), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies indicated. ( e ) Top: bacterially purified GST-USP7 was incubated with amylose resin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteins retained on the amylose resin were immunoblotted with the USP7 antibody. Bottom: bacterially purified recombinant proteins were analyzed by SDS-PAGE and Coomassie blue staining. * indicates the predicted position. ( f ) 293T cells were transfected with SFB-USP7 and treated with cycloheximide (CHX). Cells were harvested at different time points and immunoblotted with antibodies indicated. ( g, h ) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7, g ) or transduced with ZEB1 shRNA (sh-ZEB1, h ), and treated with cycloheximide. Cells were harvested at different time points and immunoblotted with antibodies indicated. ( i ) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into 293T cells. Lysates from cells with or without 6 Gy IR treatment were immunoprecipitated with the CHK1 antibody and immunoblotted with the HA antibody. Cells were treated with MG132 (10 μM) for 6 hr before harvest. ( j ) Top: ubiquitinated CHK1 was incubated with SFB-GFP control or SFB-USP7 purified with streptavidin-sepharose beads from 293T cells with or without ZEB1 co-transfection. The reaction mixture was then immunoprecipitated with the FLAG antibody and immunoblotted with the CHK1 antibody. Bottom: purified SFB-USP7 was immunoblotted with antibodies to ZEB1 and USP7. ( k ) Clonogenic survival assays of USP7 siRNA-transfected SUM159-P2 cells. n = 3 wells per group. Data in k are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transduction, shRNA, Irradiation, Immunoprecipitation, Transfection, Purification, Incubation, Recombinant, SDS Page, Staining, Cotransfection, Two Tailed Test

    ATM phosphorylates and stabilizes ZEB1 ( a ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to ATM and FLAG. ( b ) SUM159-P2 cells were transduced with ATM shRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 and GAPDH. ( c ) SUM159-P2 cells with or without Ku55933 pretreatment (10 μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml), harvested at different time points, immunoprecipitated with the ZEB1 antibody and immunoblotted with antibodies to p-S/TQ and ZEB1. ( d ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( e ) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 and SUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1. ( f ) Consensus ATM phosphorylation site on human ZEB1 (S585) and alignment with the conserved site on mouse, rat and Xenopus Zeb1. ( g ) 293T cells were transfected with wild-type, the S585A or S585D mutant of SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( h ) Immunopurified wild-type ZEB1 or the S585A mutant was incubated with immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinase buffer containing 32 P-ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to autoradiography and immunoblotting with antibodies to ZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinase activity. ( i ) HeLa cells were co-transfected with SFB-GFP and wild-type, the S585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR, harvested at different time points and immunoblotted with antibodies to FLAG. SFB-GFP serves as the control for transfection. ( j ) Clonogenic survival assays of SUM159-P0 cells transfected with wild-type ZEB1 or the mutant. n = 3 wells per group. Data in j are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ATM phosphorylates and stabilizes ZEB1 ( a ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to ATM and FLAG. ( b ) SUM159-P2 cells were transduced with ATM shRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 and GAPDH. ( c ) SUM159-P2 cells with or without Ku55933 pretreatment (10 μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml), harvested at different time points, immunoprecipitated with the ZEB1 antibody and immunoblotted with antibodies to p-S/TQ and ZEB1. ( d ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( e ) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 and SUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1. ( f ) Consensus ATM phosphorylation site on human ZEB1 (S585) and alignment with the conserved site on mouse, rat and Xenopus Zeb1. ( g ) 293T cells were transfected with wild-type, the S585A or S585D mutant of SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( h ) Immunopurified wild-type ZEB1 or the S585A mutant was incubated with immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinase buffer containing 32 P-ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to autoradiography and immunoblotting with antibodies to ZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinase activity. ( i ) HeLa cells were co-transfected with SFB-GFP and wild-type, the S585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR, harvested at different time points and immunoblotted with antibodies to FLAG. SFB-GFP serves as the control for transfection. ( j ) Clonogenic survival assays of SUM159-P0 cells transfected with wild-type ZEB1 or the mutant. n = 3 wells per group. Data in j are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transfection, Transduction, shRNA, Immunoprecipitation, Mutagenesis, Incubation, SDS Page, Autoradiography, Purification, Positive Control, Activity Assay, Two Tailed Test

    ZEB1 specifically promotes the interaction between USP7 and CHK1 ( a ) 293T cells were transfected with SFB-USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( b ) 293T cells were transfected with ZEB1 siRNA alone or in combination with SFB-USP7, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( c ) SUM159-P2 cells were transfected with ZEB1 siRNA, followed by immunoprecipitation with the USP7 antibody and immunoblotting with antibodies to CHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ZEB1 specifically promotes the interaction between USP7 and CHK1 ( a ) 293T cells were transfected with SFB-USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( b ) 293T cells were transfected with ZEB1 siRNA alone or in combination with SFB-USP7, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( c ) SUM159-P2 cells were transfected with ZEB1 siRNA, followed by immunoprecipitation with the USP7 antibody and immunoblotting with antibodies to CHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transfection, Immunoprecipitation

    14) Product Images from "Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses"

    Article Title: Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00577

    Cpd A potently inhibits transcriptional activity of human RORγt in the full-length context in T-cells. (A) Full-length RORγt drives wild-type RORE-dependent activation of a reporter gene in HUT78 T-cells expressing RORγt, but not in RORγt negative cells. HUT78 cells stably expressing RORγt or empty control vector were transfected with 4 x RORE or mutated RORE-luciferase reporter constructs, followed by stimulation with CD3 antibody/PMA for 48 h and by quantifying luciferase activity. (B) Gene expression level of ROR γ/ ROR γ t in HUT78 T-cells stably expressing RORγt or control vector together with RORE or mutated RORE-reporter genes. (C) Cpd A potently blocked RORE-mediated transcription of the luciferase reporter gene. HUT78 T-cells were stimulated with CD3 antibody plus PMA for 48 h followed by measurement of luciferase activity. (D) HUT78 T-cells stably expressing RORγt were incubated with serial dilutions of Cpd A at the beginning of the stimulation with PMA and anti-CD3 antibody and after 48 h IL-17A or IL-2 (E) concentrations were measured by ELISA. Representative examples of concentration-response curves from three independent experiments are shown. The average IC 50 value obtained for SR2211 in the RORγt-IL-17A inhibition assay was 257 ± 181 nM ( n = 2). (F) RORγt transduced HUT78 T-cells were pre-incubated with Cpd A (10 μM) or DMSO, followed by a 2 h stimulation with anti-CD3 antibody/PMA and nuclear extracts were prepared. Nuclear extracts were subjected to pull-down experiments using mutated (Mut-ROREs) or wild-type (WT ROREs) biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. Nuclear extracts (left panel) and RORE pull-down complexes (right panel) were subjected to SDS PAGE followed by RORγ Western blot analysis. Results shown are representative of three experiments with triplicate readings with similar results, except for (B) which originates from a single experiment. Error bars show the SD. Statistical analyses were performed using one way ANOVA Dunnett's test, **** p
    Figure Legend Snippet: Cpd A potently inhibits transcriptional activity of human RORγt in the full-length context in T-cells. (A) Full-length RORγt drives wild-type RORE-dependent activation of a reporter gene in HUT78 T-cells expressing RORγt, but not in RORγt negative cells. HUT78 cells stably expressing RORγt or empty control vector were transfected with 4 x RORE or mutated RORE-luciferase reporter constructs, followed by stimulation with CD3 antibody/PMA for 48 h and by quantifying luciferase activity. (B) Gene expression level of ROR γ/ ROR γ t in HUT78 T-cells stably expressing RORγt or control vector together with RORE or mutated RORE-reporter genes. (C) Cpd A potently blocked RORE-mediated transcription of the luciferase reporter gene. HUT78 T-cells were stimulated with CD3 antibody plus PMA for 48 h followed by measurement of luciferase activity. (D) HUT78 T-cells stably expressing RORγt were incubated with serial dilutions of Cpd A at the beginning of the stimulation with PMA and anti-CD3 antibody and after 48 h IL-17A or IL-2 (E) concentrations were measured by ELISA. Representative examples of concentration-response curves from three independent experiments are shown. The average IC 50 value obtained for SR2211 in the RORγt-IL-17A inhibition assay was 257 ± 181 nM ( n = 2). (F) RORγt transduced HUT78 T-cells were pre-incubated with Cpd A (10 μM) or DMSO, followed by a 2 h stimulation with anti-CD3 antibody/PMA and nuclear extracts were prepared. Nuclear extracts were subjected to pull-down experiments using mutated (Mut-ROREs) or wild-type (WT ROREs) biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. Nuclear extracts (left panel) and RORE pull-down complexes (right panel) were subjected to SDS PAGE followed by RORγ Western blot analysis. Results shown are representative of three experiments with triplicate readings with similar results, except for (B) which originates from a single experiment. Error bars show the SD. Statistical analyses were performed using one way ANOVA Dunnett's test, **** p

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Plasmid Preparation, Transfection, Luciferase, Construct, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition, SDS Page, Western Blot

    15) Product Images from "The microtubule plus-end tracking protein Bik1 is required for chromosome congression"

    Article Title: The microtubule plus-end tracking protein Bik1 is required for chromosome congression

    Journal: bioRxiv

    doi: 10.1101/2021.06.16.447861

    Bik1 interacts with Cin8 at metaphase (A-B) Identification of Cin8 as a Bik1 interaction partner using TurboID. Cartoon outlining the principle of the TurboID assay. Briefly, a biotin ligase was fused to endogenous Bik1 (Bik1-TurboID) in cells expressing the protein of interest tagged with Flag (Prey). Biotinylated substrates were purified using streptavidin beads, and inputs and pull-down lysates were analyzed by immunoblot for the indicated proteins. (B) Western blot analysis of total cell extracts (input) and streptavidin pull-downs prepared from Bik1-TurboID-3xmyc cells expressing Kip2-5xFlag or Cin8-5xFlag as prey, and control strains, using anti-Myc (top) and anti-Flag (bottom) antibodies. (*) unspecific band. (C-D) Bimolecular fluorescence complementation (BiFC) analysis of Bik1 and Cin8 interaction. (C) Representative images of cycling cells expressing Bik1-VN and Cin8-VC in G1, metaphase and anaphase. Images shown are single focal planes. (D) Proportion of cells in (C) with BIFC signal based on Venus fluorescence (3 independent experiments, n=50 cells per experiment and cell cycle stage). Error bars, SD.
    Figure Legend Snippet: Bik1 interacts with Cin8 at metaphase (A-B) Identification of Cin8 as a Bik1 interaction partner using TurboID. Cartoon outlining the principle of the TurboID assay. Briefly, a biotin ligase was fused to endogenous Bik1 (Bik1-TurboID) in cells expressing the protein of interest tagged with Flag (Prey). Biotinylated substrates were purified using streptavidin beads, and inputs and pull-down lysates were analyzed by immunoblot for the indicated proteins. (B) Western blot analysis of total cell extracts (input) and streptavidin pull-downs prepared from Bik1-TurboID-3xmyc cells expressing Kip2-5xFlag or Cin8-5xFlag as prey, and control strains, using anti-Myc (top) and anti-Flag (bottom) antibodies. (*) unspecific band. (C-D) Bimolecular fluorescence complementation (BiFC) analysis of Bik1 and Cin8 interaction. (C) Representative images of cycling cells expressing Bik1-VN and Cin8-VC in G1, metaphase and anaphase. Images shown are single focal planes. (D) Proportion of cells in (C) with BIFC signal based on Venus fluorescence (3 independent experiments, n=50 cells per experiment and cell cycle stage). Error bars, SD.

    Techniques Used: Expressing, Purification, Western Blot, Fluorescence, Bimolecular Fluorescence Complementation Assay

    16) Product Images from "CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression"

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.213868

    SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.
    Figure Legend Snippet: SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.

    Techniques Used: Purification, Mass Spectrometry, Immunoprecipitation, Expressing, Size-exclusion Chromatography, Transfection, Binding Assay

    CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.
    Figure Legend Snippet: CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Techniques Used: Plasmid Preparation, Transfection, Transduction, shRNA, Over Expression, Immunoprecipitation, Pull Down Assay, Incubation, Purification, Expressing, Recombinant, Staining, SDS Page

    17) Product Images from "Deubiquitylation Regulates Activation and Proteolytic Cleavage of ENaC"

    Article Title: Deubiquitylation Regulates Activation and Proteolytic Cleavage of ENaC

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2007101130

    Usp2-45 enhances cell surface expression of ENaC and proteolytically cleaved γENaC. HEK293 cells stably expressing αβγENaC were transfected with Usp2-45 WT or Usp2-45-C67A (Usp2-45 CA) constructs. Twenty-four hours after transfection, αENaC was induced with 1 μM dexamethasone. After another 24 h, cells were biotinylated as described in the Concise Methods section. Cells were then lysed, and the biotinylated fraction was recovered with streptavidin Sepharose. Asterisks and arrowhead indicate the 77- and 73-kD fragments for γENaC. (A) Lysates and nonbiotinylated and biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. No actin was detected in the biotinylated fraction. (B) Quantification of αβγENaC in biotinylated fraction. The levels of α, β, and γENaC were detected by Western blot fluorography and quantified on a molecular imager FX. Values were normalized to the control (ENaC alone) and displayed as mean ± SEM ( n = 3 experiments) * P
    Figure Legend Snippet: Usp2-45 enhances cell surface expression of ENaC and proteolytically cleaved γENaC. HEK293 cells stably expressing αβγENaC were transfected with Usp2-45 WT or Usp2-45-C67A (Usp2-45 CA) constructs. Twenty-four hours after transfection, αENaC was induced with 1 μM dexamethasone. After another 24 h, cells were biotinylated as described in the Concise Methods section. Cells were then lysed, and the biotinylated fraction was recovered with streptavidin Sepharose. Asterisks and arrowhead indicate the 77- and 73-kD fragments for γENaC. (A) Lysates and nonbiotinylated and biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. No actin was detected in the biotinylated fraction. (B) Quantification of αβγENaC in biotinylated fraction. The levels of α, β, and γENaC were detected by Western blot fluorography and quantified on a molecular imager FX. Values were normalized to the control (ENaC alone) and displayed as mean ± SEM ( n = 3 experiments) * P

    Techniques Used: Expressing, Stable Transfection, Transfection, Construct, SDS Page, Western Blot

    Cell surface ENaC subunits are ubiquitylated, and Usp2-45 interferes with this ubiquitylation. HEK293 cells were transiently transfected with αβγENaC together with Usp2-45, Usp2-45 CA, WT or dominant negative dynamin (DynK44R), or Nedd4-2. After 24 h of transfection, cells were biotinylated and then lysed. Immunoprecipitations were performed using antibodies against the tags of the ENaC subunits (HA for α, myc for β, and VSV for γ). Immunoprecipitated proteins were recovered, and the biotinylated fraction was precipitated with streptavidin Sepharose beads. Proteins were analyzed by SDS-PAGE/Western blotting as indicated.
    Figure Legend Snippet: Cell surface ENaC subunits are ubiquitylated, and Usp2-45 interferes with this ubiquitylation. HEK293 cells were transiently transfected with αβγENaC together with Usp2-45, Usp2-45 CA, WT or dominant negative dynamin (DynK44R), or Nedd4-2. After 24 h of transfection, cells were biotinylated and then lysed. Immunoprecipitations were performed using antibodies against the tags of the ENaC subunits (HA for α, myc for β, and VSV for γ). Immunoprecipitated proteins were recovered, and the biotinylated fraction was precipitated with streptavidin Sepharose beads. Proteins were analyzed by SDS-PAGE/Western blotting as indicated.

    Techniques Used: Transfection, Dominant Negative Mutation, Immunoprecipitation, SDS Page, Western Blot

    18) Product Images from "PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7"

    Article Title: PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

    Journal: Nature Communications

    doi: 10.1038/ncomms10689

    Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.
    Figure Legend Snippet: Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.

    Techniques Used: Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Dominant Negative Mutation, Incubation, Purification, Binding Assay, In Vitro, Recombinant, Staining

    PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.
    Figure Legend Snippet: PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.

    Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Expressing, Mutagenesis, Construct

    19) Product Images from "PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7"

    Article Title: PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

    Journal: Nature Communications

    doi: 10.1038/ncomms10689

    Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.
    Figure Legend Snippet: Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.

    Techniques Used: Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Dominant Negative Mutation, Incubation, Purification, Binding Assay, In Vitro, Recombinant, Staining

    PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.
    Figure Legend Snippet: PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.

    Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Expressing, Mutagenesis, Construct

    20) Product Images from "Molecular Mechanism of Cell-autonomous Circadian Gene Expression of Period2, a Crucial Regulator of the Mammalian Circadian Clock D⃞"

    Article Title: Molecular Mechanism of Cell-autonomous Circadian Gene Expression of Period2, a Crucial Regulator of the Mammalian Circadian Clock D⃞

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E05-05-0396

    A potential mechanism by which the cell-autonomous core clock generates more overt circadian oscillations in Per2 transcription than in Per1 transcription. (A and B) Temporal expression patterns of the mPer1 and mPer2 genes, in mouse peripheral tissues (A) and in serum-stimulated NIH3T3 cells (B) were assayed by real-time quantitative RT-PCR. Mice were kept in a 12-h light:12-h dark cycle (LD, lights on 8 a.m.; lights off 8 p.m.) for 2 wk to establish entrainment. Three animals were killed at the times given on the abscissas of the diagrams. Each value represents the average of three independent RT-PCR experiments. The relative levels of each RNA were normalized to the corresponding Gapdh RNA levels. Peak values of the mPer1 and mPer2 curves were set to 1. (C) Transcriptional oscillation of mPer2 -luc, mPer2 -luc (E-box), and mPer1 -luc was monitored in real time. NIH3T3 cells were transfected and then stimulated with a high concentration of serum. Peak values of the curves were set to 1 (vertical scale: relative cpm; horizontal scale: 1440 min = 1 d). A representative result of three independent experiments is shown. (D and E) The signals obtained in C were detrended. (F) COS7 cells were transfected with the E4BP4-luciferase fusion expression vector. Cell extracts were incubated with the double-stranded biotinylated Per2 (-45 to +15) oligonucleotide, including the consensus-predicted E4BP4 response elements, which was immobilized on streptavidin-Sepharose beads. The negative control samples were treated with streptavidin-Sepharose beads without an oligonucleotide. The resulting precipitates were subjected to luciferase assays. Data represent the mean ± SEM of triplicate samples. (G) Transcriptional oscillation of Per2 (-105)-luc was monitored, in the presence or absence of E4BP4 (left). The signals obtained were detrended (right). (H) With increasing dose of the E4BP4 expression plasmid, transcriptional oscillation of Per2 (-105)-luc was monitored. The periods were obtained from analysis of a circadian marker. Data represent the mean ± SEM of triplicate samples. (I) Schematic model representing BMAL1:CLOCK-mediated control of cell-autonomous Per1 and Per2 oscillation. A combination of the E-box-like sequence and a cooperating element increases the amplitude of rhythmic transcription of Per2 , and consequently the amplitude of Per2 mRNA rhythms is significantly higher than that of Per1 mRNA rhythms. The 1-base pairs difference (CACGTT) from the classical circadian E-box may be indispensable for this combination. The cell-autonomous core clock generates overt circadian oscillations in Per2 transcription, whereas the high amplitude of Per1 oscillation in vivo largely depends on the extracellular environment, which changes cyclically around the clock, rather than on the core clock. Thus, the Per1 gene might not be tightly incorporated into the cell-autonomous core clock mechanism.
    Figure Legend Snippet: A potential mechanism by which the cell-autonomous core clock generates more overt circadian oscillations in Per2 transcription than in Per1 transcription. (A and B) Temporal expression patterns of the mPer1 and mPer2 genes, in mouse peripheral tissues (A) and in serum-stimulated NIH3T3 cells (B) were assayed by real-time quantitative RT-PCR. Mice were kept in a 12-h light:12-h dark cycle (LD, lights on 8 a.m.; lights off 8 p.m.) for 2 wk to establish entrainment. Three animals were killed at the times given on the abscissas of the diagrams. Each value represents the average of three independent RT-PCR experiments. The relative levels of each RNA were normalized to the corresponding Gapdh RNA levels. Peak values of the mPer1 and mPer2 curves were set to 1. (C) Transcriptional oscillation of mPer2 -luc, mPer2 -luc (E-box), and mPer1 -luc was monitored in real time. NIH3T3 cells were transfected and then stimulated with a high concentration of serum. Peak values of the curves were set to 1 (vertical scale: relative cpm; horizontal scale: 1440 min = 1 d). A representative result of three independent experiments is shown. (D and E) The signals obtained in C were detrended. (F) COS7 cells were transfected with the E4BP4-luciferase fusion expression vector. Cell extracts were incubated with the double-stranded biotinylated Per2 (-45 to +15) oligonucleotide, including the consensus-predicted E4BP4 response elements, which was immobilized on streptavidin-Sepharose beads. The negative control samples were treated with streptavidin-Sepharose beads without an oligonucleotide. The resulting precipitates were subjected to luciferase assays. Data represent the mean ± SEM of triplicate samples. (G) Transcriptional oscillation of Per2 (-105)-luc was monitored, in the presence or absence of E4BP4 (left). The signals obtained were detrended (right). (H) With increasing dose of the E4BP4 expression plasmid, transcriptional oscillation of Per2 (-105)-luc was monitored. The periods were obtained from analysis of a circadian marker. Data represent the mean ± SEM of triplicate samples. (I) Schematic model representing BMAL1:CLOCK-mediated control of cell-autonomous Per1 and Per2 oscillation. A combination of the E-box-like sequence and a cooperating element increases the amplitude of rhythmic transcription of Per2 , and consequently the amplitude of Per2 mRNA rhythms is significantly higher than that of Per1 mRNA rhythms. The 1-base pairs difference (CACGTT) from the classical circadian E-box may be indispensable for this combination. The cell-autonomous core clock generates overt circadian oscillations in Per2 transcription, whereas the high amplitude of Per1 oscillation in vivo largely depends on the extracellular environment, which changes cyclically around the clock, rather than on the core clock. Thus, the Per1 gene might not be tightly incorporated into the cell-autonomous core clock mechanism.

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Concentration Assay, Luciferase, Plasmid Preparation, Incubation, Negative Control, Marker, Sequencing, In Vivo

    Temporal patterns of the site-specific binding of endogenous circadian transcription factors to the Per2 E-box-like sequence. Mouse liver extracts were harvested at 4-h intervals and then subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Mouse liver extracts were incubated with a double-stranded biotinylated oligonucleotide including the consensus-predicted Per2 E-box-like sequence (CACGTT) or three different Per1 E-boxes (wild-type, WT; mutant, MT), which was immobilized on streptavidin-Sepharose beads. The negative control samples were treated with the beads without an oligonucleotide (No ODN). The resulting precipitates were subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Temporal expression patterns of the mPer2 and mPer1 genes in mouse liver were assayed by real-time quantitative RT-PCR (bottom panels). Each value represents the average of three independent RT-PCR experiments. The relative levels were normalized to the corresponding Gapdh RNA levels. Peak values of the mPer2 and mPer1 curves were set to 1.
    Figure Legend Snippet: Temporal patterns of the site-specific binding of endogenous circadian transcription factors to the Per2 E-box-like sequence. Mouse liver extracts were harvested at 4-h intervals and then subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Mouse liver extracts were incubated with a double-stranded biotinylated oligonucleotide including the consensus-predicted Per2 E-box-like sequence (CACGTT) or three different Per1 E-boxes (wild-type, WT; mutant, MT), which was immobilized on streptavidin-Sepharose beads. The negative control samples were treated with the beads without an oligonucleotide (No ODN). The resulting precipitates were subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Temporal expression patterns of the mPer2 and mPer1 genes in mouse liver were assayed by real-time quantitative RT-PCR (bottom panels). Each value represents the average of three independent RT-PCR experiments. The relative levels were normalized to the corresponding Gapdh RNA levels. Peak values of the mPer2 and mPer1 curves were set to 1.

    Techniques Used: Binding Assay, Sequencing, Incubation, Mutagenesis, Negative Control, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    21) Product Images from "Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes"

    Article Title: Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2770

    Biochemical validation of asymmetric nucleosome formation in vivo A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.
    Figure Legend Snippet: Biochemical validation of asymmetric nucleosome formation in vivo A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.

    Techniques Used: In Vivo, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Flow Cytometry, SDS Page

    22) Product Images from "WWP2-WWP1 Ubiquitin Ligase Complex Coordinated by PPM1G Maintains the Balance between Cellular p73 and ΔNp73 Levels"

    Article Title: WWP2-WWP1 Ubiquitin Ligase Complex Coordinated by PPM1G Maintains the Balance between Cellular p73 and ΔNp73 Levels

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00101-14

    p73 is a WWP2-associated protein. (a) 293T cells were transfected with either Flag-tagged p73 or SFB-tagged p53 together with Myc-tagged WWP2. Immunoprecipitation was performed using anti-IgG or anti-Flag antibody, followed by immunoblotting with anti-Myc antibody. (b) 293T cells were separately transfected with Flag-p73α, Flag-p73β, Flag-ΔNp73α, and SFB-p53 together with Myc-WWP2. Immunoprecipitation was performed using anti-Flag followed by immunoblotting with anti-Myc antibody. (c) Bacterial cell lysate expressing MBP-WWP2 was added to GST, GST-p73, or GST-ΔNp73 immobilized on agarose beads. The in vitro interaction of WWP2 was assessed by immunoblotting with MBP antibody. The expression of GST, GST-p73, GST-ΔNp73, and MBP-WWP2 was shown by Coomassie staining. (d) The indicated mutants of p73 were coexpressed along with WWP2, and their interaction was determined by immunoblotting with Myc antibody after immunoprecipitation using streptavidin beads (SBP). α, anti; IP, immunoprecipitation; WB, Western blotting.
    Figure Legend Snippet: p73 is a WWP2-associated protein. (a) 293T cells were transfected with either Flag-tagged p73 or SFB-tagged p53 together with Myc-tagged WWP2. Immunoprecipitation was performed using anti-IgG or anti-Flag antibody, followed by immunoblotting with anti-Myc antibody. (b) 293T cells were separately transfected with Flag-p73α, Flag-p73β, Flag-ΔNp73α, and SFB-p53 together with Myc-WWP2. Immunoprecipitation was performed using anti-Flag followed by immunoblotting with anti-Myc antibody. (c) Bacterial cell lysate expressing MBP-WWP2 was added to GST, GST-p73, or GST-ΔNp73 immobilized on agarose beads. The in vitro interaction of WWP2 was assessed by immunoblotting with MBP antibody. The expression of GST, GST-p73, GST-ΔNp73, and MBP-WWP2 was shown by Coomassie staining. (d) The indicated mutants of p73 were coexpressed along with WWP2, and their interaction was determined by immunoblotting with Myc antibody after immunoprecipitation using streptavidin beads (SBP). α, anti; IP, immunoprecipitation; WB, Western blotting.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, In Vitro, Staining, Western Blot

    23) Product Images from "ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1"

    Article Title: ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1

    Journal: Nature cell biology

    doi: 10.1038/ncb3013

    ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1 ( a ) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10 μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates were immunoprecipitated with the CHK1 antibody and immunoblotted with antibodies indicated. ( b ) A partial list of ZEB1-associated proteins. ( c, d ) 293T cells were transfected with SFB-ZEB1 ( c ) or SFB-USP7 ( d ), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies indicated. ( e ) Top: bacterially purified GST-USP7 was incubated with amylose resin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteins retained on the amylose resin were immunoblotted with the USP7 antibody. Bottom: bacterially purified recombinant proteins were analyzed by SDS-PAGE and Coomassie blue staining. * indicates the predicted position. ( f ) 293T cells were transfected with SFB-USP7 and treated with cycloheximide (CHX). Cells were harvested at different time points and immunoblotted with antibodies indicated. ( g, h ) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7, g ) or transduced with ZEB1 shRNA (sh-ZEB1, h ), and treated with cycloheximide. Cells were harvested at different time points and immunoblotted with antibodies indicated. ( i ) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into 293T cells. Lysates from cells with or without 6 Gy IR treatment were immunoprecipitated with the CHK1 antibody and immunoblotted with the HA antibody. Cells were treated with MG132 (10 μM) for 6 hr before harvest. ( j ) Top: ubiquitinated CHK1 was incubated with SFB-GFP control or SFB-USP7 purified with streptavidin-sepharose beads from 293T cells with or without ZEB1 co-transfection. The reaction mixture was then immunoprecipitated with the FLAG antibody and immunoblotted with the CHK1 antibody. Bottom: purified SFB-USP7 was immunoblotted with antibodies to ZEB1 and USP7. ( k ) Clonogenic survival assays of USP7 siRNA-transfected SUM159-P2 cells. n = 3 wells per group. Data in k are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ZEB1 interacts with USP7 which deubiquitinates and stabilizes CHK1 ( a ) SUM159-P2 cells transduced with ZEB1 shRNA were treated with 10 μM MG132, irradiated with 6 Gy IR and harvested 6 hr later. Lysates were immunoprecipitated with the CHK1 antibody and immunoblotted with antibodies indicated. ( b ) A partial list of ZEB1-associated proteins. ( c, d ) 293T cells were transfected with SFB-ZEB1 ( c ) or SFB-USP7 ( d ), followed by pull-down with streptavidin-sepharose beads (s-s beads) and immunoblotting with antibodies indicated. ( e ) Top: bacterially purified GST-USP7 was incubated with amylose resin conjugated with bacterially expressed MBP-GFP or MBP-ZEB1. Proteins retained on the amylose resin were immunoblotted with the USP7 antibody. Bottom: bacterially purified recombinant proteins were analyzed by SDS-PAGE and Coomassie blue staining. * indicates the predicted position. ( f ) 293T cells were transfected with SFB-USP7 and treated with cycloheximide (CHX). Cells were harvested at different time points and immunoblotted with antibodies indicated. ( g, h ) SUM159-P2 cells were transfected with USP7 siRNA (si-USP7, g ) or transduced with ZEB1 shRNA (sh-ZEB1, h ), and treated with cycloheximide. Cells were harvested at different time points and immunoblotted with antibodies indicated. ( i ) HA-ubiquitin was co-transfected with SFB-GFP or SFB-USP7 into 293T cells. Lysates from cells with or without 6 Gy IR treatment were immunoprecipitated with the CHK1 antibody and immunoblotted with the HA antibody. Cells were treated with MG132 (10 μM) for 6 hr before harvest. ( j ) Top: ubiquitinated CHK1 was incubated with SFB-GFP control or SFB-USP7 purified with streptavidin-sepharose beads from 293T cells with or without ZEB1 co-transfection. The reaction mixture was then immunoprecipitated with the FLAG antibody and immunoblotted with the CHK1 antibody. Bottom: purified SFB-USP7 was immunoblotted with antibodies to ZEB1 and USP7. ( k ) Clonogenic survival assays of USP7 siRNA-transfected SUM159-P2 cells. n = 3 wells per group. Data in k are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transduction, shRNA, Irradiation, Immunoprecipitation, Transfection, Purification, Incubation, Recombinant, SDS Page, Staining, Cotransfection, Two Tailed Test

    ATM phosphorylates and stabilizes ZEB1 ( a ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to ATM and FLAG. ( b ) SUM159-P2 cells were transduced with ATM shRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 and GAPDH. ( c ) SUM159-P2 cells with or without Ku55933 pretreatment (10 μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml), harvested at different time points, immunoprecipitated with the ZEB1 antibody and immunoblotted with antibodies to p-S/TQ and ZEB1. ( d ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( e ) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 and SUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1. ( f ) Consensus ATM phosphorylation site on human ZEB1 (S585) and alignment with the conserved site on mouse, rat and Xenopus Zeb1. ( g ) 293T cells were transfected with wild-type, the S585A or S585D mutant of SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( h ) Immunopurified wild-type ZEB1 or the S585A mutant was incubated with immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinase buffer containing 32 P-ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to autoradiography and immunoblotting with antibodies to ZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinase activity. ( i ) HeLa cells were co-transfected with SFB-GFP and wild-type, the S585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR, harvested at different time points and immunoblotted with antibodies to FLAG. SFB-GFP serves as the control for transfection. ( j ) Clonogenic survival assays of SUM159-P0 cells transfected with wild-type ZEB1 or the mutant. n = 3 wells per group. Data in j are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ATM phosphorylates and stabilizes ZEB1 ( a ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to ATM and FLAG. ( b ) SUM159-P2 cells were transduced with ATM shRNA and treated with IR. Lysates were immunoblotted with antibodies to p-ATM, ATM, ZEB1, CHK1 and GAPDH. ( c ) SUM159-P2 cells with or without Ku55933 pretreatment (10 μM, 1 hr) were treated with IR (6 Gy) and CHX (50 μg/ml), harvested at different time points, immunoprecipitated with the ZEB1 antibody and immunoblotted with antibodies to p-S/TQ and ZEB1. ( d ) 293T cells were transfected with SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( e ) Endogenous ZEB1 was immunoprecipitated from SUM159-P0 and SUM159-P2 cells and immunoblotted with antibodies to p-S/TQ and ZEB1. ( f ) Consensus ATM phosphorylation site on human ZEB1 (S585) and alignment with the conserved site on mouse, rat and Xenopus Zeb1. ( g ) 293T cells were transfected with wild-type, the S585A or S585D mutant of SFB-ZEB1 and treated with IR, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to p-S/TQ and ZEB1. ( h ) Immunopurified wild-type ZEB1 or the S585A mutant was incubated with immunopurified wild-type ATM or the kinase-dead (KD) mutant in kinase buffer containing 32 P-ATP. After reaction, proteins were resolved by SDS-PAGE and subjected to autoradiography and immunoblotting with antibodies to ZEB1 and p-ATM. Purified GST-p53 was used as a positive control for ATM kinase activity. ( i ) HeLa cells were co-transfected with SFB-GFP and wild-type, the S585A or S585D mutant of SFB-ZEB1, treated with CHX with or without IR, harvested at different time points and immunoblotted with antibodies to FLAG. SFB-GFP serves as the control for transfection. ( j ) Clonogenic survival assays of SUM159-P0 cells transfected with wild-type ZEB1 or the mutant. n = 3 wells per group. Data in j are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t -test. The experiments were repeated 3 times. The source data can be found in Supplementary Table 3 . Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transfection, Transduction, shRNA, Immunoprecipitation, Mutagenesis, Incubation, SDS Page, Autoradiography, Purification, Positive Control, Activity Assay, Two Tailed Test

    ZEB1 specifically promotes the interaction between USP7 and CHK1 ( a ) 293T cells were transfected with SFB-USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( b ) 293T cells were transfected with ZEB1 siRNA alone or in combination with SFB-USP7, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( c ) SUM159-P2 cells were transfected with ZEB1 siRNA, followed by immunoprecipitation with the USP7 antibody and immunoblotting with antibodies to CHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7 .
    Figure Legend Snippet: ZEB1 specifically promotes the interaction between USP7 and CHK1 ( a ) 293T cells were transfected with SFB-USP7 alone or in combination with ZEB1, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( b ) 293T cells were transfected with ZEB1 siRNA alone or in combination with SFB-USP7, followed by pull-down with streptavidin-sepharose beads and immunoblotting with antibodies to CHK1, HLTF, p53 and USP7. ( c ) SUM159-P2 cells were transfected with ZEB1 siRNA, followed by immunoprecipitation with the USP7 antibody and immunoblotting with antibodies to CHK1 and USP7. Uncropped images of blots are shown in Supplementary Figure 7 .

    Techniques Used: Transfection, Immunoprecipitation

    24) Product Images from "Potential contribution of tandem circadian enhancers to nonlinear oscillations in clock gene expression"

    Article Title: Potential contribution of tandem circadian enhancers to nonlinear oscillations in clock gene expression

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-02-0129

    The hCry2 tandem E-boxes interact directly with BMAL1 and CLOCK in an interdependent manner. (A) Nucleic acid sequences of a region of hCry2 containing E1 and E2. Asterisks show conserved nucleotides among human, rat, and mouse. The E-boxes are indicated by red squares. Sequences of mutant oligonucleotides used for EMSA and pull-down experiments are shown as mE1, mE2, and mE1 + mE2. A 32 P-labeled, double-stranded oligonucleotide that contained the hCry2 tandem E-boxes was used as an EMSA probe. The purified BMAL1 and CLOCK complex was incubated with the probe. The BMAL1 and CLOCK–bound probe is indicated as BM-CL-probe. The complex was shifted by addition of specific antibodies (Super shift). (B) Liver extracts were incubated with double-stranded biotinylated oligonucleotides ( hCry2-ODN ), which were immobilized on streptavidin-Sepharose beads. Negative control samples were incubated with streptavidin-Sepharose beads without oligonucleotides (Unconjugated). The resulting precipitates were subjected to immunoblot analysis with anti-BMAL1 and anti-CLOCK antibodies. (C) Competitor experiments. The purified BMAL1 and CLOCK complex was incubated with the labeled probe containing the hCry2 tandem E-boxes in the presence of unlabeled probes. The amount of the unlabeled probes is indicated as relative levels to the labeled hCry2 tandem E-box probe. Middle, autoradiography signals quantified with Typhoon FLA9500. Data are representative of more than two independent experiments.
    Figure Legend Snippet: The hCry2 tandem E-boxes interact directly with BMAL1 and CLOCK in an interdependent manner. (A) Nucleic acid sequences of a region of hCry2 containing E1 and E2. Asterisks show conserved nucleotides among human, rat, and mouse. The E-boxes are indicated by red squares. Sequences of mutant oligonucleotides used for EMSA and pull-down experiments are shown as mE1, mE2, and mE1 + mE2. A 32 P-labeled, double-stranded oligonucleotide that contained the hCry2 tandem E-boxes was used as an EMSA probe. The purified BMAL1 and CLOCK complex was incubated with the probe. The BMAL1 and CLOCK–bound probe is indicated as BM-CL-probe. The complex was shifted by addition of specific antibodies (Super shift). (B) Liver extracts were incubated with double-stranded biotinylated oligonucleotides ( hCry2-ODN ), which were immobilized on streptavidin-Sepharose beads. Negative control samples were incubated with streptavidin-Sepharose beads without oligonucleotides (Unconjugated). The resulting precipitates were subjected to immunoblot analysis with anti-BMAL1 and anti-CLOCK antibodies. (C) Competitor experiments. The purified BMAL1 and CLOCK complex was incubated with the labeled probe containing the hCry2 tandem E-boxes in the presence of unlabeled probes. The amount of the unlabeled probes is indicated as relative levels to the labeled hCry2 tandem E-box probe. Middle, autoradiography signals quantified with Typhoon FLA9500. Data are representative of more than two independent experiments.

    Techniques Used: Mutagenesis, Labeling, Purification, Incubation, Negative Control, Autoradiography

    25) Product Images from "Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania"

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007237

    Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.
    Figure Legend Snippet: Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.

    Techniques Used: Transgenic Assay, Expressing, Fractionation, SDS Page, Migration, Western Blot, Flow Cytometry

    The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.
    Figure Legend Snippet: The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.

    Techniques Used: Mutagenesis, Migration, SDS Page, Transgenic Assay, Expressing, Western Blot, Copurification, Flow Cytometry, Confocal Microscopy, Incubation, Labeling, Staining

    Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p
    Figure Legend Snippet: Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Luciferase, Software

    26) Product Images from "A Public Health Antibody Screening Indicates a 6-Fold Higher SARS-CoV-2 Exposure Rate than Reported Cases in Children"

    Article Title: A Public Health Antibody Screening Indicates a 6-Fold Higher SARS-CoV-2 Exposure Rate than Reported Cases in Children

    Journal: Med (New York, N.y.)

    doi: 10.1016/j.medj.2020.10.003

    Serum Inhibition of RBD Binding to Its Receptor ACE2 (A) The ability of SARS-CoV-2 antibody-negative sera (open circles, n = 22 children) and -positive sera (filled green circles, n = 74 children) to inhibit the binding of nanoluciferase-tagged RBD to biotinylated recombinant ACE2 coated streptavidin Sepharose beads. Maximum RBD binding to ACE2-Sepharose beads corresponded to approximately 90,000 light units and background binding of RBD to uncoated beads corresponded to approximately 300 light units. (B) Inhibition of binding (y axis) is shown against the SARS-CoV-2 RBD antibody titer (x axis) for the antibody-positive children (n = 74).
    Figure Legend Snippet: Serum Inhibition of RBD Binding to Its Receptor ACE2 (A) The ability of SARS-CoV-2 antibody-negative sera (open circles, n = 22 children) and -positive sera (filled green circles, n = 74 children) to inhibit the binding of nanoluciferase-tagged RBD to biotinylated recombinant ACE2 coated streptavidin Sepharose beads. Maximum RBD binding to ACE2-Sepharose beads corresponded to approximately 90,000 light units and background binding of RBD to uncoated beads corresponded to approximately 300 light units. (B) Inhibition of binding (y axis) is shown against the SARS-CoV-2 RBD antibody titer (x axis) for the antibody-positive children (n = 74).

    Techniques Used: Inhibition, Binding Assay, Recombinant

    27) Product Images from "Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach"

    Article Title: Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01409-9

    The scheme for Met-peptide enrichment. Reduced Met-peptides are labeled by the iodoacetyl-PEG2-biotin reagent under acidic conditions, whereas the oxidized Met-peptides are not. The labeled peptides are then captured and purified with streptavidin-Sepharose beads. After reduction by DTT, the captured Met-peptides are released from the beads and analyzed by LC-MS/MS.
    Figure Legend Snippet: The scheme for Met-peptide enrichment. Reduced Met-peptides are labeled by the iodoacetyl-PEG2-biotin reagent under acidic conditions, whereas the oxidized Met-peptides are not. The labeled peptides are then captured and purified with streptavidin-Sepharose beads. After reduction by DTT, the captured Met-peptides are released from the beads and analyzed by LC-MS/MS.

    Techniques Used: Labeling, Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    28) Product Images from "Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)- > Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3"

    Article Title: Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)- > Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3

    Journal:

    doi: 10.1158/0008-5472.CAN-07-3036

    CDDO-Me binds directly to STAT3 at Cys 259 . A , HeLa cell lysates were incubated with 1 μmol/L of biotin or CDDO-Me-biotin for 2 h. Streptavidin-Sepharose beads were then added for 1 h. The precipitates were immunoblotted with anti-STAT3 ( left ).
    Figure Legend Snippet: CDDO-Me binds directly to STAT3 at Cys 259 . A , HeLa cell lysates were incubated with 1 μmol/L of biotin or CDDO-Me-biotin for 2 h. Streptavidin-Sepharose beads were then added for 1 h. The precipitates were immunoblotted with anti-STAT3 ( left ).

    Techniques Used: Incubation

    29) Product Images from "PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7"

    Article Title: PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

    Journal: Nature Communications

    doi: 10.1038/ncomms10689

    Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.
    Figure Legend Snippet: Rab7 is PTEN-associated protein. ( a ) HEK 293T cells transfected with the SFB-tagged Rab7 or ( b ) with SFB-tagged PTEN construct was subjected to immunoprecipitation (IP) with either control IgG or Flag antibody and the interaction of endogenous PTEN and Rab7 was determined by western blotting (WB) with their specific antibodies, respectively. ( c ) 293T cells were transfected with triple-tagged SFB-Rab5 or SFB-Rab7 and their interaction with PTEN was detected by immunoblotting with PTEN-specific antibody after immunoprecipitating with streptavidin (SBP) beads. ( d ) HEK293T cell lysates expressing SFB Rab7 WT or T22N (a dominant negative GDP bound) and Q67L (constitutively GTP-bound active) mutants were incubated with bacterially purified GST PTEN. The association of PTEN with Rab7 and its mutants was detected by immunoblotting with PTEN antibody. ( e ) SFB-Rab7 expressing HEK293T cell lysates pre-loaded either with GDPβS or GTPγS was incubated with glutathione–sepharose bound bacterially purified GST-PTEN and their binding was analysed by western blotting with Flag antibody. ( f ) GST Rab7 was loaded w ith either GTPγS or GDPβS in vitro . The association of PTEN with Rab7 was detected by immunoblotting with PTEN antibody after passing the 293T cell lysate through pre-loaded recombinant Rab7. ( g ) Agarose beads immobilized with bacterially expressed recombinant MBP-PTEN was incubated with either GST or GST-Rab7 proteins expressed in bacteria in the presence of GDP. The direct association of Rab7 with PTEN was detected by immunoblotting with GST antibody. Expression of the recombinant GST Rab7 and MBP-PTEN was shown by coomassie staining. ( h ) Schematic representation of N-terminal Myc-tagged PTEN (FL), along with its deletion mutants (D1–D4). ( i ) Myc-tagged PTEN constructs and SFB-Rab7 were co-expressed in HEK 293T cells, and the interaction of PTEN with Rab7 was detected by immunoblotting with anti-Myc antibodies after the cell lysates were pulled down with streptavidin beads.

    Techniques Used: Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Dominant Negative Mutation, Incubation, Purification, Binding Assay, In Vitro, Recombinant, Staining

    PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.
    Figure Legend Snippet: PTEN-mediated Rab7 dephosphorylation is necessary for its interaction with GDI, GEF and effector proteins. ( a ) HEK 293T cells transfected with either SFB-Rab7 WT or its various mutants were subjected to immunoprecipitation with streptavidin beads (SBP). The interaction of GDI with Rab7 and its mutants was analysed by immunoblotting with GDI-specific antibody. ( b ) Cells were transfected with SFB-Rab7 WT and its mutants along with Flag-tagged Ccz1 and the interaction of Rab7 with Ccz1 was determined by immunoblotting with Flag antibody after immunoprecipitating with SBP. ( c ) 293T cells expressing either control or two different PTEN shRNAs were co-transfected with SFB-Rab7 and Flag-Ccz1. The interaction of Ccz1 with Rab7 was analysed by immunoblotting with Flag antibody after immunoprecipitation with SBP beads. ( d ) Cells were transfected with SFB-Rab7 WT and its mutants along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( e ) Cells were transfected with SFB-Rab7 WT and Rab7 S72A/Y183D mutant along with GFP-tagged RILP and the interaction of Rab7 with RILP was determined by immunoblotting with GFP antibody after immunoprecipitating with SBP. ( f ) Cells transfected with various indicated SFB-tagged Rab7 constructs were labelled with 32 P-orthophosphate. GDP and GTP levels were analysed by thin layer chromotagraphy after immunoprecipitating Rab7 from the cell lysates by using streptravidin sepharose. ( g ) The GTP/GDP-bound ratio of various Rab7 mutants quantified by using Phosphorimager was plotted.

    Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Expressing, Mutagenesis, Construct

    30) Product Images from "CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression"

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.213868

    SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.
    Figure Legend Snippet: SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.

    Techniques Used: Purification, Mass Spectrometry, Immunoprecipitation, Expressing, Size-exclusion Chromatography, Transfection, Binding Assay

    CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.
    Figure Legend Snippet: CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Techniques Used: Plasmid Preparation, Transfection, Transduction, shRNA, Over Expression, Immunoprecipitation, Pull Down Assay, Incubation, Purification, Expressing, Recombinant, Staining, SDS Page

    31) Product Images from "Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania"

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007237

    Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.
    Figure Legend Snippet: Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.

    Techniques Used: Transgenic Assay, Expressing, Fractionation, SDS Page, Migration, Western Blot, Flow Cytometry

    The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.
    Figure Legend Snippet: The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.

    Techniques Used: Mutagenesis, Migration, SDS Page, Transgenic Assay, Expressing, Western Blot, Copurification, Flow Cytometry, Confocal Microscopy, Incubation, Labeling, Staining

    Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p
    Figure Legend Snippet: Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Luciferase, Software

    32) Product Images from "Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes"

    Article Title: Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2770

    Biochemical validation of asymmetric nucleosome formation in vivo A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.
    Figure Legend Snippet: Biochemical validation of asymmetric nucleosome formation in vivo A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.

    Techniques Used: In Vivo, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Flow Cytometry, SDS Page

    33) Product Images from "Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)- > Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3"

    Article Title: Triterpenoid CDDO-Methyl Ester Inhibits the Janus-Activated Kinase-1 (JAK1)- > Signal Transducer and Activator of Transcription-3 (STAT3) Pathway by Direct Inhibition of JAK1 and STAT3

    Journal:

    doi: 10.1158/0008-5472.CAN-07-3036

    CDDO-Me binds directly to STAT3 at Cys 259 . A , HeLa cell lysates were incubated with 1 μmol/L of biotin or CDDO-Me-biotin for 2 h. Streptavidin-Sepharose beads were then added for 1 h. The precipitates were immunoblotted with anti-STAT3 ( left ).
    Figure Legend Snippet: CDDO-Me binds directly to STAT3 at Cys 259 . A , HeLa cell lysates were incubated with 1 μmol/L of biotin or CDDO-Me-biotin for 2 h. Streptavidin-Sepharose beads were then added for 1 h. The precipitates were immunoblotted with anti-STAT3 ( left ).

    Techniques Used: Incubation

    34) Product Images from "Novel inhibitors of a Grb2 SH3C domain interaction identified by a virtual screen"

    Article Title: Novel inhibitors of a Grb2 SH3C domain interaction identified by a virtual screen

    Journal: Bioorganic & medicinal chemistry

    doi: 10.1016/j.bmc.2012.10.023

    Grb2 SH3C competitive binding screen with 34 hit molecules using a biochemical competition assay. Purified GST–Grb2 SH3C was incubated with each compound (5 mM) in duplicate before contact with streptavidin-sepharose beads previously coupled to
    Figure Legend Snippet: Grb2 SH3C competitive binding screen with 34 hit molecules using a biochemical competition assay. Purified GST–Grb2 SH3C was incubated with each compound (5 mM) in duplicate before contact with streptavidin-sepharose beads previously coupled to

    Techniques Used: Binding Assay, Competitive Binding Assay, Purification, Incubation

    35) Product Images from "CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression"

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.213868

    SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.
    Figure Legend Snippet: SMU1 assembles CRL type E3 ligase complex by interacting with DDB1, CUL7 and RNF40. (A) Proteins that contain a LisH domain and WD repeats. (B) Partial list of SMU1-associated proteins identified by biochemical purification followed by MS analysis were listed together with the number of peptides for each protein. (C) Immunoprecipitation (IP) with control IgG or anti-SMU1 antibody was performed with extracts prepared from HEK-293T cells. The presence of RNF40, DDB1, CUL7 and DYRK2 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (D) SFB-tagged VPRBP, together with either Myc-tagged SMU1 or Myc-tagged DYRK2, was expressed in cells and the interaction of the respective proteins was detected by immunoblotting with the indicated antibodies after pulling down the complexes with streptavidin Sepharose. (E,F) HA tagged-SKP1 together with either SFB-tagged SMU1 or SFB-tagged ROC1 (E), and SFB-tagged ROC1 together with Myc-tagged SMU1 or HA-tagged SKP1 (F) were expressed in cells and their interaction was detected as described in D. (G) HeLa cells expressing Myc-tagged RNF20 were lysed and immunoprecipitation was carried out using either IgG or anti-Myc antibody. The presence of SMU1 and RNF40 was detected in these immunoprecipitates by immunoblotting using specific antibodies. (H) HEK-293T cell extracts were analysed by size-exclusion chromatography using a Sephacryl 300 column. Proteins eluted from the different fractions were immunoblotted with antibodies against the indicated proteins. (I) Domain architecture of full-length SMU1 (SMU1 FL) and its deletion mutants. (J) SFB-tagged SMU1 FL and SMU1 deletion mutants were transfected in HeLa cells. 24 h post transfection, cells were lysed and pull-down was carried out using Streptavidin-binding peptide (SBP) beads. The presence of DDB1, CUL7 and RNF40 in these precipitates was evaluated by immunoblotting with their respective antibodies.

    Techniques Used: Purification, Immunoprecipitation, Expressing, Size-exclusion Chromatography, Transfection, Binding Assay

    CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.
    Figure Legend Snippet: CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Techniques Used: Plasmid Preparation, Transfection, Transduction, shRNA, Over Expression, Immunoprecipitation, Pull Down Assay, Incubation, Purification, Expressing, Recombinant, Staining, SDS Page

    36) Product Images from "Androgen Activation of the Folate Receptor ? Gene through Partial Tethering of the Androgen Receptor by C/EBP? ○"

    Article Title: Androgen Activation of the Folate Receptor ? Gene through Partial Tethering of the Androgen Receptor by C/EBP? ○

    Journal: The Journal of steroid biochemistry and molecular biology

    doi: 10.1016/j.jsbmb.2010.08.008

    Physical interaction between AR and C/EBPα and recruitment of endogenous AR to the FRα gene (A) DNA pull down assays for biotinylated forms of a synthetic FRα promoter element (-1570nt to-1533nt) or 3-tandem repeat C/EBP elements [(C/EBP) 3 ]: Hela cells were tranfected with AR plasmid for 48h and treated with testosterone (10 nM) or vehicle for 1h before being harvested to prepare total cell lysates. The lysates were incubated with each biotinylated probe in the continued presence of either testosterone or vehicle. Control assays included incubation with a 200-fold excess of unlabeled synthetic DNA corresponding to FRα, -1570nt to-1533nt (wild type probe, wt); the wt probe in which the ARE half-site was mutated (mA); the wt probe in which both the ARE half-site and the C/EBP element were mutated (dM). The biotinylated DNA probe and associated proteins were pulled down using streptavidin sepharose beads. The proteins were eluted from the beads and probed by western blot analysis using antibody to AR or C/EBPα. (B) Co-immunoprecipitation of AR and C/EBPα in ACH-3P cells: Endogenous AR in the cell lysates was immonoprecipitated and probed by western blot using antibodies to either AR or C/EBPα. Bands were detected using either antibody probe in the immunoprecipitate obtained using anti-AR antibody but not in the negative control. (C) Chromatin immunoprecipitation of AR in R1881 treated ACH-3P cells. The control target sequence (irrelevant target) corresponds to the estrogen receptor gene. The FRα target sequence corresponds to the -1565nt to -1534nt region.
    Figure Legend Snippet: Physical interaction between AR and C/EBPα and recruitment of endogenous AR to the FRα gene (A) DNA pull down assays for biotinylated forms of a synthetic FRα promoter element (-1570nt to-1533nt) or 3-tandem repeat C/EBP elements [(C/EBP) 3 ]: Hela cells were tranfected with AR plasmid for 48h and treated with testosterone (10 nM) or vehicle for 1h before being harvested to prepare total cell lysates. The lysates were incubated with each biotinylated probe in the continued presence of either testosterone or vehicle. Control assays included incubation with a 200-fold excess of unlabeled synthetic DNA corresponding to FRα, -1570nt to-1533nt (wild type probe, wt); the wt probe in which the ARE half-site was mutated (mA); the wt probe in which both the ARE half-site and the C/EBP element were mutated (dM). The biotinylated DNA probe and associated proteins were pulled down using streptavidin sepharose beads. The proteins were eluted from the beads and probed by western blot analysis using antibody to AR or C/EBPα. (B) Co-immunoprecipitation of AR and C/EBPα in ACH-3P cells: Endogenous AR in the cell lysates was immonoprecipitated and probed by western blot using antibodies to either AR or C/EBPα. Bands were detected using either antibody probe in the immunoprecipitate obtained using anti-AR antibody but not in the negative control. (C) Chromatin immunoprecipitation of AR in R1881 treated ACH-3P cells. The control target sequence (irrelevant target) corresponds to the estrogen receptor gene. The FRα target sequence corresponds to the -1565nt to -1534nt region.

    Techniques Used: Plasmid Preparation, Incubation, Western Blot, Immunoprecipitation, Negative Control, Chromatin Immunoprecipitation, Sequencing

    37) Product Images from "Expansion of DUB functionality generated by alternative isoforms – USP35, a case study"

    Article Title: Expansion of DUB functionality generated by alternative isoforms – USP35, a case study

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.212753

    Isoform-specific interactome of USP35. (A) HEK293 FlpIn cells expressing USP35 iso1 or USP35 iso2 tagged at their C-terminus with BirA R118G were induced for 24 h. Biotinylated proteins were recovered on streptavidin–Sepharose, identified by mass spectrometry and subjected to SAINT analysis and DAVID GO analysis. Green bars show GO terms for the percentage of genes identified; red lines indicates the −log 10 P value. (B) Isoform-specific and shared interacting partners are shown.
    Figure Legend Snippet: Isoform-specific interactome of USP35. (A) HEK293 FlpIn cells expressing USP35 iso1 or USP35 iso2 tagged at their C-terminus with BirA R118G were induced for 24 h. Biotinylated proteins were recovered on streptavidin–Sepharose, identified by mass spectrometry and subjected to SAINT analysis and DAVID GO analysis. Green bars show GO terms for the percentage of genes identified; red lines indicates the −log 10 P value. (B) Isoform-specific and shared interacting partners are shown.

    Techniques Used: Expressing, Mass Spectrometry

    38) Product Images from "A newly identified Leishmania IF4E-interacting protein, Leish4E-IP2, modulates the activity of cap-binding protein paralogs"

    Article Title: A newly identified Leishmania IF4E-interacting protein, Leish4E-IP2, modulates the activity of cap-binding protein paralogs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa173

    Association of Leish4E-IP2 with different LeishIF4Es. The association between Leish4E-IP2 and LeishIF4E-1 (4E1), LeishIF4E-4 (4E4) ( A ) and LeishIF4E-3 (4E3) ( B ) were monitored by pull-down experiments of mid-log L. amazonensis promastigotes over-expressing SBP-tagged LeishIF4E-IP2. The cells were lysed and affinity-purified over streptavidin-Sepharose beads. The beads were washed and further eluted with biotin. Aliquots from the soluble extract (S, 5%), the flow-through fraction (FT, 5%), the final wash (W, 50%) and eluted proteins (E, 50%) were separated by 10% SDS–PAGE and subjected to western blot analysis using specific antibodies. The antibodies used were raised against LeishIF4E-1, LeishIF4E-4 (A), LeishIF4E-3 and LeishIF4A (as a negative control) (B) along with anti-SBP antibodies that were used to highlight the SBP-tagged Leish4E-IP2. ( C ) Representation of the densitometry analysis of each quantity of LeishIF4E co-eluted by Leish4E-IP2.
    Figure Legend Snippet: Association of Leish4E-IP2 with different LeishIF4Es. The association between Leish4E-IP2 and LeishIF4E-1 (4E1), LeishIF4E-4 (4E4) ( A ) and LeishIF4E-3 (4E3) ( B ) were monitored by pull-down experiments of mid-log L. amazonensis promastigotes over-expressing SBP-tagged LeishIF4E-IP2. The cells were lysed and affinity-purified over streptavidin-Sepharose beads. The beads were washed and further eluted with biotin. Aliquots from the soluble extract (S, 5%), the flow-through fraction (FT, 5%), the final wash (W, 50%) and eluted proteins (E, 50%) were separated by 10% SDS–PAGE and subjected to western blot analysis using specific antibodies. The antibodies used were raised against LeishIF4E-1, LeishIF4E-4 (A), LeishIF4E-3 and LeishIF4A (as a negative control) (B) along with anti-SBP antibodies that were used to highlight the SBP-tagged Leish4E-IP2. ( C ) Representation of the densitometry analysis of each quantity of LeishIF4E co-eluted by Leish4E-IP2.

    Techniques Used: Expressing, Affinity Purification, SDS Page, Western Blot, Negative Control

    39) Product Images from "Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses"

    Article Title: Antagonizing Retinoic Acid-Related-Orphan Receptor Gamma Activity Blocks the T Helper 17/Interleukin-17 Pathway Leading to Attenuated Pro-inflammatory Human Keratinocyte and Skin Responses

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00577

    Cpd A potently inhibits transcriptional activity of human RORγt in the full-length context in T-cells. (A) Full-length RORγt drives wild-type RORE-dependent activation of a reporter gene in HUT78 T-cells expressing RORγt, but not in RORγt negative cells. HUT78 cells stably expressing RORγt or empty control vector were transfected with 4 x RORE or mutated RORE-luciferase reporter constructs, followed by stimulation with CD3 antibody/PMA for 48 h and by quantifying luciferase activity. (B) Gene expression level of ROR γ/ ROR γ t in HUT78 T-cells stably expressing RORγt or control vector together with RORE or mutated RORE-reporter genes. (C) Cpd A potently blocked RORE-mediated transcription of the luciferase reporter gene. HUT78 T-cells were stimulated with CD3 antibody plus PMA for 48 h followed by measurement of luciferase activity. (D) HUT78 T-cells stably expressing RORγt were incubated with serial dilutions of Cpd A at the beginning of the stimulation with PMA and anti-CD3 antibody and after 48 h IL-17A or IL-2 (E) concentrations were measured by ELISA. Representative examples of concentration-response curves from three independent experiments are shown. The average IC 50 value obtained for SR2211 in the RORγt-IL-17A inhibition assay was 257 ± 181 nM ( n = 2). (F) RORγt transduced HUT78 T-cells were pre-incubated with Cpd A (10 μM) or DMSO, followed by a 2 h stimulation with anti-CD3 antibody/PMA and nuclear extracts were prepared. Nuclear extracts were subjected to pull-down experiments using mutated (Mut-ROREs) or wild-type (WT ROREs) biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. Nuclear extracts (left panel) and RORE pull-down complexes (right panel) were subjected to SDS PAGE followed by RORγ Western blot analysis. Results shown are representative of three experiments with triplicate readings with similar results, except for (B) which originates from a single experiment. Error bars show the SD. Statistical analyses were performed using one way ANOVA Dunnett's test, **** p
    Figure Legend Snippet: Cpd A potently inhibits transcriptional activity of human RORγt in the full-length context in T-cells. (A) Full-length RORγt drives wild-type RORE-dependent activation of a reporter gene in HUT78 T-cells expressing RORγt, but not in RORγt negative cells. HUT78 cells stably expressing RORγt or empty control vector were transfected with 4 x RORE or mutated RORE-luciferase reporter constructs, followed by stimulation with CD3 antibody/PMA for 48 h and by quantifying luciferase activity. (B) Gene expression level of ROR γ/ ROR γ t in HUT78 T-cells stably expressing RORγt or control vector together with RORE or mutated RORE-reporter genes. (C) Cpd A potently blocked RORE-mediated transcription of the luciferase reporter gene. HUT78 T-cells were stimulated with CD3 antibody plus PMA for 48 h followed by measurement of luciferase activity. (D) HUT78 T-cells stably expressing RORγt were incubated with serial dilutions of Cpd A at the beginning of the stimulation with PMA and anti-CD3 antibody and after 48 h IL-17A or IL-2 (E) concentrations were measured by ELISA. Representative examples of concentration-response curves from three independent experiments are shown. The average IC 50 value obtained for SR2211 in the RORγt-IL-17A inhibition assay was 257 ± 181 nM ( n = 2). (F) RORγt transduced HUT78 T-cells were pre-incubated with Cpd A (10 μM) or DMSO, followed by a 2 h stimulation with anti-CD3 antibody/PMA and nuclear extracts were prepared. Nuclear extracts were subjected to pull-down experiments using mutated (Mut-ROREs) or wild-type (WT ROREs) biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. Nuclear extracts (left panel) and RORE pull-down complexes (right panel) were subjected to SDS PAGE followed by RORγ Western blot analysis. Results shown are representative of three experiments with triplicate readings with similar results, except for (B) which originates from a single experiment. Error bars show the SD. Statistical analyses were performed using one way ANOVA Dunnett's test, **** p

    Techniques Used: Activity Assay, Activation Assay, Expressing, Stable Transfection, Plasmid Preparation, Transfection, Luciferase, Construct, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition, SDS Page, Western Blot

    40) Product Images from "Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo"

    Article Title: Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0188391

    Cpd 1 blocks IL17A / IL23R gene expression by inhibiting permissive chromatin remodeling at their promoter regions. (A) RORγt or empty vector transduced HUT78 T-cells were stimulated with anti-CD3 antibody and PMA. IL17A and IL23R gene expression was analyzed by RT-PCR, which was performed on mRNA isolated after 24 hrs of stimulation. (B) RORγt transduced HUT78 T-cells were stimulated as described above in the presence of cpd 1 (10 μM) or DMSO and mRNA was prepared followed by analysis of IL17A , IL23R and RORC gene expression via RT-PCR. Gene expression levels are shown relative to DMSO treated cells (100%). (C and D) Stimulated cell lysates from cpd 1(10 μM) or DMSO-treated RORγt transduced HUT78 T-cells were cross-linked with 1% formaldehyde, sonicated and chromatin preparations were immunoprecipitated with H3K9/14 acetyl or H3K4me3-specific or isotype-specific control antibodies. The precipitated DNA was quantified by qPCR with primers specific for IL17A and IL23R promoter regions. The results were normalized to an input control. (E) RORγt transduced HUT78 T-cells were treated with cpd 1(10 μM ) or DMSO, stimulated with anti-CD3 antibody/PMA or left unstimulated (No stim) for 2 hrs and nuclear extracts were prepared. Nuclear extracts were equally divided into two and each half was subjected to pull-down experiments using mutated or wild-type biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. After extensive washing pull-down complexes (left panel) or nuclear extracts (right panel) were subjected to SDS PAGE and RORγ Western blot analysis. Graphs and Western blots are representative of two independent experiments.
    Figure Legend Snippet: Cpd 1 blocks IL17A / IL23R gene expression by inhibiting permissive chromatin remodeling at their promoter regions. (A) RORγt or empty vector transduced HUT78 T-cells were stimulated with anti-CD3 antibody and PMA. IL17A and IL23R gene expression was analyzed by RT-PCR, which was performed on mRNA isolated after 24 hrs of stimulation. (B) RORγt transduced HUT78 T-cells were stimulated as described above in the presence of cpd 1 (10 μM) or DMSO and mRNA was prepared followed by analysis of IL17A , IL23R and RORC gene expression via RT-PCR. Gene expression levels are shown relative to DMSO treated cells (100%). (C and D) Stimulated cell lysates from cpd 1(10 μM) or DMSO-treated RORγt transduced HUT78 T-cells were cross-linked with 1% formaldehyde, sonicated and chromatin preparations were immunoprecipitated with H3K9/14 acetyl or H3K4me3-specific or isotype-specific control antibodies. The precipitated DNA was quantified by qPCR with primers specific for IL17A and IL23R promoter regions. The results were normalized to an input control. (E) RORγt transduced HUT78 T-cells were treated with cpd 1(10 μM ) or DMSO, stimulated with anti-CD3 antibody/PMA or left unstimulated (No stim) for 2 hrs and nuclear extracts were prepared. Nuclear extracts were equally divided into two and each half was subjected to pull-down experiments using mutated or wild-type biotinylated RORE oligonucleotides followed by immobilization of complexes with streptavidin Sepharose beads. After extensive washing pull-down complexes (left panel) or nuclear extracts (right panel) were subjected to SDS PAGE and RORγ Western blot analysis. Graphs and Western blots are representative of two independent experiments.

    Techniques Used: Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Isolation, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, SDS Page, Western Blot

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    Article Snippet: Cells were lysed in 0.5% Nonidet P-40 Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5) with protease inhibitor (Roche) and PMSF (Sigma) freshly added following the manufacturer’s guide. .. Immunoprecipitation was carried out by sequentially incubating Anti-Flag M2 Affinity Gel (Sigma) and Streptavidin Sepharose (GE Healthcare) with cell lysate for 3 to 4 h at 4 °C. .. Precipitated proteins were washed with Tris buffer three times and we competitively eluted Anti-Flag M2 Affinity Gel by 3×Flag peptides (Sigma) and Streptavidin Sepharose by biotin (Sigma), respectively.

    Competitive Binding Assay:

    Article Title: SBF-1 exerts strong anticervical cancer effect through inducing endoplasmic reticulum stress-associated cell death via targeting sarco/endoplasmic reticulum Ca2+-ATPase 2
    Article Snippet: .. Competitive binding assay HeLa whole-cell lysates were, respectively, incubated with 10 μ M biotin, 10 μ M biotin-SBF-1, 10 μ M biotin-SBF-1 plus 100 μ M SBF-1 or 10 μ M biotin-SBF-1 plus 200 μ M SBF-1 and 50 μ l streptavidin-conjugated sepharose beads (GE Healthcare) at 4 °C overnight for 12 h. Then, the mixture was centrifuged at 4000 × g for 1 min to obtain the precipitation. .. After washing five times with RIPA lysis buffer, the beads were boiled in 2 × loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 1% bromphenol blue, 20% glycerol and 2% β -mercaptoethanol).

    Incubation:

    Article Title: SBF-1 exerts strong anticervical cancer effect through inducing endoplasmic reticulum stress-associated cell death via targeting sarco/endoplasmic reticulum Ca2+-ATPase 2
    Article Snippet: .. Competitive binding assay HeLa whole-cell lysates were, respectively, incubated with 10 μ M biotin, 10 μ M biotin-SBF-1, 10 μ M biotin-SBF-1 plus 100 μ M SBF-1 or 10 μ M biotin-SBF-1 plus 200 μ M SBF-1 and 50 μ l streptavidin-conjugated sepharose beads (GE Healthcare) at 4 °C overnight for 12 h. Then, the mixture was centrifuged at 4000 × g for 1 min to obtain the precipitation. .. After washing five times with RIPA lysis buffer, the beads were boiled in 2 × loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 1% bromphenol blue, 20% glycerol and 2% β -mercaptoethanol).

    Article Title: Identification of FUSE-binding protein 1 as a regulatory mRNA-binding protein that represses nucleophosmin translation
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    Article Title: The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿
    Article Snippet: These beads were incubated with B. abortus 2308 whole lysates or a suspension of purified BvrR, and the pulled-down proteins were analyzed by Western blotting. .. BvrR was detected in the pulldown experiment when the lysate was incubated with streptavidin-Sepharose beads coated with the VirB promoter region; in contrast, BvrR was absent when the lysate was incubated with streptavidin-Sepharose beads loaded with a biotinylated amplicon comprising the promoter region of the operon dhbCEBA (negative control) ( ) (Fig. ). .. The biotinylated VirB probe, but not the dhbCEBA probe, was able to bind VjbR (positive control) in this pulldown experiment, thus confirming the approach (Fig. ).

    other:

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania
    Article Snippet: Fractions 25–42 were pooled and subjected to pull-down analysis using streptavidin-Sepharose beads.

    Amplification:

    Article Title: The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿
    Article Snippet: These beads were incubated with B. abortus 2308 whole lysates or a suspension of purified BvrR, and the pulled-down proteins were analyzed by Western blotting. .. BvrR was detected in the pulldown experiment when the lysate was incubated with streptavidin-Sepharose beads coated with the VirB promoter region; in contrast, BvrR was absent when the lysate was incubated with streptavidin-Sepharose beads loaded with a biotinylated amplicon comprising the promoter region of the operon dhbCEBA (negative control) ( ) (Fig. ). .. The biotinylated VirB probe, but not the dhbCEBA probe, was able to bind VjbR (positive control) in this pulldown experiment, thus confirming the approach (Fig. ).

    Negative Control:

    Article Title: The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿
    Article Snippet: These beads were incubated with B. abortus 2308 whole lysates or a suspension of purified BvrR, and the pulled-down proteins were analyzed by Western blotting. .. BvrR was detected in the pulldown experiment when the lysate was incubated with streptavidin-Sepharose beads coated with the VirB promoter region; in contrast, BvrR was absent when the lysate was incubated with streptavidin-Sepharose beads loaded with a biotinylated amplicon comprising the promoter region of the operon dhbCEBA (negative control) ( ) (Fig. ). .. The biotinylated VirB probe, but not the dhbCEBA probe, was able to bind VjbR (positive control) in this pulldown experiment, thus confirming the approach (Fig. ).

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    WD4- and WD6-peptides reduce CFTR surface expression. WD peptides were delivered into Calu-3 cells using BioPORTER reagent. The apical membrane was biotinylated, and biotinylated proteins were recovered by streptavidin pulldown. Proteins were probed for

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    Article Title: RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

    doi: 10.1152/ajpcell.00026.2013

    Figure Lengend Snippet: WD4- and WD6-peptides reduce CFTR surface expression. WD peptides were delivered into Calu-3 cells using BioPORTER reagent. The apical membrane was biotinylated, and biotinylated proteins were recovered by streptavidin pulldown. Proteins were probed for

    Article Snippet: Cells were harvested in CFTR lysis buffer, and biotinylated proteins were isolated using streptavidin-agarose beads (GE Healthcare), eluted into SDS sample buffer supplemented with 50 mM dithiothreitol, and separated by SDS-PAGE.

    Techniques: Expressing

    ENaC ubiquitylation in whole cell lysates and at the cell surface. A , Hek293 cells were transiently transfected with either wild-type or KR mutant ENaC channels. The ubiquitylated and the total amount of ENaC expressed in Hek293 cells were visualized by Western blotting of immunoprecipitated α-(HA), β-(c-Myc), and γ-(VSV)-ENaC, with either anti ubiquitin or anti ENaC antibodies. B , cells were biotinylated, lysed and then immunoprecipitated with HA tag (for α), c-Myc tag (for β), and VSV tag (for γ). The channels present at the cells surface were recovered using streptavidin-Sepharose beads. Biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. ENaC KR : K to R mutation on all cytoplasmic lysines. fu : mutation on furin sites. The nature of a fragment seen occasionally at ∼70 kDa in the ENaC blots is not known (*). C , Hek293 cells were transiently transfected with ENaC WT +/− Nedd4-2. Cells were biotinylated 24 h after transfection. Then they were lysed and an immunoprecipitation was performed with anti-HA to recover αENaC. The cell surface channels were recovered using streptavidin beads. Then proteins were run on SDS/PAGE and Western blot were performed as describe. Quantification of three individual experiments of the ratio ubiquitylated/full-length αENaC was calculated. They were normalized to αENaC full-length and displayed as mean ± S.E. ( n = 3 experiments, *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change *

    doi: 10.1074/jbc.M110.176156

    Figure Lengend Snippet: ENaC ubiquitylation in whole cell lysates and at the cell surface. A , Hek293 cells were transiently transfected with either wild-type or KR mutant ENaC channels. The ubiquitylated and the total amount of ENaC expressed in Hek293 cells were visualized by Western blotting of immunoprecipitated α-(HA), β-(c-Myc), and γ-(VSV)-ENaC, with either anti ubiquitin or anti ENaC antibodies. B , cells were biotinylated, lysed and then immunoprecipitated with HA tag (for α), c-Myc tag (for β), and VSV tag (for γ). The channels present at the cells surface were recovered using streptavidin-Sepharose beads. Biotinylated proteins were analyzed by SDS-PAGE/Western blotting as indicated. ENaC KR : K to R mutation on all cytoplasmic lysines. fu : mutation on furin sites. The nature of a fragment seen occasionally at ∼70 kDa in the ENaC blots is not known (*). C , Hek293 cells were transiently transfected with ENaC WT +/− Nedd4-2. Cells were biotinylated 24 h after transfection. Then they were lysed and an immunoprecipitation was performed with anti-HA to recover αENaC. The cell surface channels were recovered using streptavidin beads. Then proteins were run on SDS/PAGE and Western blot were performed as describe. Quantification of three individual experiments of the ratio ubiquitylated/full-length αENaC was calculated. They were normalized to αENaC full-length and displayed as mean ± S.E. ( n = 3 experiments, *, p

    Article Snippet: Supernatants were recovered, and we added 900 μl of PBS and 30 μl streptavidin-Sepharose beads (GE Healthcare).

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

    Proteolytic cleavage of wild-type and mutant channels mutated on cytoplasmic lysines. A , Hek293 cells were transiently transfected with either wild-type ( W ) or cytoplasmic lysine mutant ( K ) ENaC. 24 h after transfection, cells were biotinylated, recovered with streptavidin-Sepharose and analyzed by SDS-PAGE/Western blotting using anti α-, or γ-ENaC antibodies as indicated. fl : full-length α- or γ-ENaC; arrow : cleaved α- or γ-ENaC. α- and γENaC antibodies crossreact with endogenous proteins, as shown in lane 1 . In our previous work we have provided evidence that these do not represent endogenous ENaC. B , quantification of the ratio of cleaved to full-length αENaC (as described under “Experimental Procedures”), normalized to wild-type ENaC (condition 2), and displayed as mean ± S.E. ( n = 3 experiments; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Intracellular Ubiquitylation of the Epithelial Na+ Channel Controls Extracellular Proteolytic Channel Activation via Conformational Change *

    doi: 10.1074/jbc.M110.176156

    Figure Lengend Snippet: Proteolytic cleavage of wild-type and mutant channels mutated on cytoplasmic lysines. A , Hek293 cells were transiently transfected with either wild-type ( W ) or cytoplasmic lysine mutant ( K ) ENaC. 24 h after transfection, cells were biotinylated, recovered with streptavidin-Sepharose and analyzed by SDS-PAGE/Western blotting using anti α-, or γ-ENaC antibodies as indicated. fl : full-length α- or γ-ENaC; arrow : cleaved α- or γ-ENaC. α- and γENaC antibodies crossreact with endogenous proteins, as shown in lane 1 . In our previous work we have provided evidence that these do not represent endogenous ENaC. B , quantification of the ratio of cleaved to full-length αENaC (as described under “Experimental Procedures”), normalized to wild-type ENaC (condition 2), and displayed as mean ± S.E. ( n = 3 experiments; *, p

    Article Snippet: Supernatants were recovered, and we added 900 μl of PBS and 30 μl streptavidin-Sepharose beads (GE Healthcare).

    Techniques: Mutagenesis, Transfection, SDS Page, Western Blot

    Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    doi: 10.1371/journal.pntd.0007237

    Figure Lengend Snippet: Enrichment of starvation-induced LeishIF4E-3-containing granules over sucrose gradients. Transgenic L . amazonensis promastigotes expressing SBP-tagged LeishIF4E-3 were fully starved (PBS, right panel) or kept under normal conditions as controls (left panel). (A) Cell extracts were treated with cycloheximide (100 μg/ml) followed by fractionation over 10–40% sucrose gradients. The OD 260 of the sucrose fractions is shown in the top panels. (B) Samples from the fractionated proteins were precipitated by TCA and further resolved over 12% SDS-PAGE. The migration profile of the proteins was shown by western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with 15 μl from the total supernatant fraction (S, 0.75%) and 15 μl from each fraction (fraction number, 5%). Fractions 25–42 were pooled, and further pulled-down over streptavidin-Sepharose beads. The eluted complexes were analyzed in western blots using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with a sample of the pooled fractions prior to the pull down (S, 10%) and from the flow through fraction (FT, 10%), followed by a sample of the last wash (W, 50%) and the eluted fraction (E, 50%). Similar results were obtained from three independent experiments.

    Article Snippet: Fractions 25–42 were pooled and subjected to pull-down analysis using streptavidin-Sepharose beads.

    Techniques: Transgenic Assay, Expressing, Fractionation, SDS Page, Migration, Western Blot, Flow Cytometry

    The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    doi: 10.1371/journal.pntd.0007237

    Figure Lengend Snippet: The S75A mutation of LeishIF4E3 leads to a decrease in granule formation in response to PBS starvation and to a reduced interaction with LeishIF4G-4. (A) Migration profile of the endogenous and tagged LeishIF4E-3 on SDS-PAGE under non-starved and starved conditions. Transgenic L . amazonensis promastigotes expressing either SBP-tagged LeishIF4E-3 or the S75A SBP-tagged mutant LeishIF4E3 were grown in complete DMEM or in nutrient-free buffer (PBS) for 4 h. Total cellular extracts were resolved on reduced bis-acrylamide SDS-PAGE and subjected to western analysis using specific antibodies against LeishIF4E-3, or against SBP tag. A non-starved parasite culture was used as control. (B) Co-purification of LeishIF4G-4 with SBP-tagged LeishIF4E-3 and S75A mutant LeishIF4E-3 under normal conditions. Non-starved parasites expressing either SBP-tagged LeishIF4E-3 or the S75A mutant LeishIF4E-3 were subjected to pull-down analysis over streptavidin-Sepharose beads. The eluted complexes were separated over 12% SDS-PAGE that were further subjected to western analysis using specific antibodies against LeishIF4E-3 or LeishIF4G-4. The gels were loaded with samples taken from the total supernatant prior to the pull down (S, 2%), the flow through fraction (FT, 2%), the final wash (W, 50%) and the eluted fraction (E, 50%). (C) Confocal analysis of SBP-tagged LeishIF4E-3 (I), or SBP-tagged S75A mutant LeishIF4E3 ((II), starved or non-starved. The cells were fixed, permeabilized and processed for confocal microscopy. LeishIF4E-3 was detected using rabbit anti-LeishIF4E-3 antibodies followed by incubation with anti-rabbit DyLight-labeled secondary antibodies (550 nm; red). Mutant SBP-tagged S75A LeishIF4E-3 was visualized using mouse monoclonal antibodies against SBP followed by incubation with anti-mouse DyLight-labeled secondary antibodies (488 nm; green). Nuclear and kinetoplast DNA was stained using DAPI (blue). Bright field pictures are shown on the right.

    Article Snippet: Fractions 25–42 were pooled and subjected to pull-down analysis using streptavidin-Sepharose beads.

    Techniques: Mutagenesis, Migration, SDS Page, Transgenic Assay, Expressing, Western Blot, Copurification, Flow Cytometry, Confocal Microscopy, Incubation, Labeling, Staining

    Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

    doi: 10.1371/journal.pntd.0007237

    Figure Lengend Snippet: Categorized proteomic content of the starvation-induced LeishIF4E-3 containing granules. The proteomic content of starvation-induced LeishIF4E-3 containing granules enriched over sucrose gradients and further pulled-down over streptavidin-Sepharose beads was determined by LC-MS/MS analysis, in triplicates and compared to a control pull down with a non-relevant protein. Parallel control cells expressing SBP-tagged luciferase were treated similarly and subjected to LC-MS/MS analysis, in triplicates and in the same run. The proteins were identified by the MaxQuant software using TriTrypDB database annotations. Differences between the proteomic contents of the LeishIF4E-3 and luciferase pulled-down fractions were determined using the Perseus statistical tool. Proteins enriched two fold with a p

    Article Snippet: Fractions 25–42 were pooled and subjected to pull-down analysis using streptavidin-Sepharose beads.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Expressing, Luciferase, Software

    Direct interaction of the VirB promoter with BvrR. (A) Purified BvrR protein or whole-bacterium lysates of B. abortus 2308 were incubated with streptavidin-Sepharose beads preloaded with a biotin-labeled amplicon comprising the virB promoter region or

    Journal: Journal of Bacteriology

    Article Title: The Two-Component System BvrR/BvrS Regulates the Expression of the Type IV Secretion System VirB in Brucella abortus ▿

    doi: 10.1128/JB.00567-10

    Figure Lengend Snippet: Direct interaction of the VirB promoter with BvrR. (A) Purified BvrR protein or whole-bacterium lysates of B. abortus 2308 were incubated with streptavidin-Sepharose beads preloaded with a biotin-labeled amplicon comprising the virB promoter region or

    Article Snippet: BvrR was detected in the pulldown experiment when the lysate was incubated with streptavidin-Sepharose beads coated with the VirB promoter region; in contrast, BvrR was absent when the lysate was incubated with streptavidin-Sepharose beads loaded with a biotinylated amplicon comprising the promoter region of the operon dhbCEBA (negative control) ( ) (Fig. ).

    Techniques: Purification, Incubation, Labeling, Amplification