streptavidin agarose beads  (GE Healthcare)

 
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    Streptavidin Horseradish Peroxidase
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    Structured Review

    GE Healthcare streptavidin agarose beads
    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 <t>streptavidin</t> pulldown. Proteins were probed for

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    Images

    1) Product Images from "RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator"

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

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00026.2013

    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
    Figure Legend 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

    Techniques Used: Expressing

    2) Product Images from "APEX2‐mediated RAB proximity labeling identifies a role for RAB21 in clathrin‐independent cargo sorting"

    Article Title: APEX2‐mediated RAB proximity labeling identifies a role for RAB21 in clathrin‐independent cargo sorting

    Journal: EMBO Reports

    doi: 10.15252/embr.201847192

    APEX2:RAB expression lead to endosomal biotinylation Diagram representing APEX2:RAB‐mediated endosomal biotinylation of endogenous proteins. Illustrative representation of APEX2:RAB endosomal microdomains. APEX2 only or APEX2:RAB were found to biotinylate endogenous proteins. Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp‐In/T‐REx HeLa cells. APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n = 2 independent experiments. Scale bars: 10 μm or 5 μm in enlarged views.
    Figure Legend Snippet: APEX2:RAB expression lead to endosomal biotinylation Diagram representing APEX2:RAB‐mediated endosomal biotinylation of endogenous proteins. Illustrative representation of APEX2:RAB endosomal microdomains. APEX2 only or APEX2:RAB were found to biotinylate endogenous proteins. Streptavidin Western blotting of total biotinylated proteins in APEX2:RAB or APEX2 only Flp‐In/T‐REx HeLa cells. APEX2:RAB biotinylated proteins (streptavidin) are partially colocalized with EEA1 in HeLa cells, n = 2 independent experiments. Scale bars: 10 μm or 5 μm in enlarged views.

    Techniques Used: Expressing, Western Blot

    3) Product Images from "ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)"

    Article Title: ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-10-1463

    Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.
    Figure Legend Snippet: Coimmunoprecipitation of DDR1 with ADAM10. (A) HEK293 cells were cotransfected with DDR1-NF, wild-type ADAM10, or ADAM10ΔMP in combination as indicated and subjected to immunoprecipitation with anti-FLAG affinity beads. Bound materials were analyzed by Western blotting using anti–DDR1 ectodomain or anti-ADAM10 antibodies. Asterisks indicate the protein that was pulled down. MP, metalloproteinase domain. (B) HEK293 cells were transiently cotransfected with FLAG-tagged ADAM10 (ADAM10-F), ADAM10ΔMP-F, or DDR1 in combination as indicated. Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by Western blotting with anti-DDR1 ectodomain or anti-ADAM10 antibodies. DDR1 was coimmunoprecipitated with ADAM10-F or ADAM10ΔMP-F (FLAG-immunoprecipitation, top). Asterisks indicate the protein that was pulled down. (C) Coimmunoprecipitation of endogenous DDR1 and ADAM10. A431 cells were subjected to cell surface biotinylation before cell lysis. Cell lysates were subjected to two-step affinity precipitation using antixDDR1 ectodomain or anti-ADAM10 ectodomain conjugated to protein G–coated Dynabeads followed by streptavidin beads. Coimmunoprecipitated DDR1 and ADAM10 bound to streptavidin beads were visualized by Western blotting using anti-DDR1 C-terminus or anti-ADAM10 antibodies. Control sample was incubated with protein G–Dynabeads without antibodies. Active, active form of ADAM10; Pro, proform. Blank lanes that were cropped out of the blot are indicated by black lines.

    Techniques Used: Immunoprecipitation, Western Blot, Lysis, Affinity Precipitation, Incubation

    4) Product Images from "Gfi-1B Promoter Remains Associated with Active Chromatin Marks Throughout Erythroid Differentiation of Human Primary Progenitor Cells"

    Article Title: Gfi-1B Promoter Remains Associated with Active Chromatin Marks Throughout Erythroid Differentiation of Human Primary Progenitor Cells

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.151

    LSD1 and CoREST bind to c-myc but not to Gfi-1B oligonucleotides. ( A): Gfi and GATA protein expression during erythroid differentiation of UT-7 5.3 cells. UT-7 5.3 cells cultured in the presence of granulocyte-macrophage colony-stimulating factor were induced to differentiate in the presence of erythropoietin (EPO). Cells were lysed the first day (E0) and 5 days (E5) after induction of erythroid differentiation by EPO. The nuclear cell lysates were analyzed by Western blot with Gfi-1, Gfi-1B, GATA-1 and GATA-2 specific antibodies. Actin was detected by a specific antibody to confirm equal protein loading. Cells were stained with benzidine to reveal hemoglobin and the percentage of benzidine positive cells was determined. ( B): Binding of GATA and Gfi proteins with Gfi-1B promoter oligonucleotides before and after erythroid differentiation. DNA affinity precipitation experiment with biotinylated oligonucleotides representing the Gfi-1B promoter was performed using nuclear extracts from UT-7 5.3 cells cultured in the presence of GM-CSF (E0) or stimulated by EPO during 5 days (E5). Then 4 μg of wild-type (1) or mutated (2) biotin-labeled oligonucleotides representing the Gfi-1B core of the Gfi-1B promoter (-69 to –37 from the start site) were used. The three Gfi-1/Gfi-1B binding sites in the Gfi-1B core oligonucleotide were mutated into GGTC. Proteins bound to the DNA template were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting using antibodies as indicated. ( C): Comparison of GATA-1, Gfi-1B, LSD1, and CoREST binding with Gfi-1B and c-myc oligonucleotides. DNA-binding affinity precipitation experiment was performed with biotinylated-oligonucleotides representing Gfi-1B (Gfi-1B core as above) or c-myc promoter (sequence from -868 to -840 of c-myc gene). UT-7 cells were induced to differentiate and lysates were prepared 5 days after induction of differentiation with EPO. Nuclear extracts were incubated with increasing quantities of oligonucleotides (from 1–4 μg indicated by black wedges) of wild-type or with 4 μg of mutated (indicated by a cross) oligonucleotides, precipitated with streptavidin beads and analyzed by Western blotting using antibodies against Gfi-1B or GATA-1, LSD1 and CoREST. This experiment is representative of three experiments.
    Figure Legend Snippet: LSD1 and CoREST bind to c-myc but not to Gfi-1B oligonucleotides. ( A): Gfi and GATA protein expression during erythroid differentiation of UT-7 5.3 cells. UT-7 5.3 cells cultured in the presence of granulocyte-macrophage colony-stimulating factor were induced to differentiate in the presence of erythropoietin (EPO). Cells were lysed the first day (E0) and 5 days (E5) after induction of erythroid differentiation by EPO. The nuclear cell lysates were analyzed by Western blot with Gfi-1, Gfi-1B, GATA-1 and GATA-2 specific antibodies. Actin was detected by a specific antibody to confirm equal protein loading. Cells were stained with benzidine to reveal hemoglobin and the percentage of benzidine positive cells was determined. ( B): Binding of GATA and Gfi proteins with Gfi-1B promoter oligonucleotides before and after erythroid differentiation. DNA affinity precipitation experiment with biotinylated oligonucleotides representing the Gfi-1B promoter was performed using nuclear extracts from UT-7 5.3 cells cultured in the presence of GM-CSF (E0) or stimulated by EPO during 5 days (E5). Then 4 μg of wild-type (1) or mutated (2) biotin-labeled oligonucleotides representing the Gfi-1B core of the Gfi-1B promoter (-69 to –37 from the start site) were used. The three Gfi-1/Gfi-1B binding sites in the Gfi-1B core oligonucleotide were mutated into GGTC. Proteins bound to the DNA template were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting using antibodies as indicated. ( C): Comparison of GATA-1, Gfi-1B, LSD1, and CoREST binding with Gfi-1B and c-myc oligonucleotides. DNA-binding affinity precipitation experiment was performed with biotinylated-oligonucleotides representing Gfi-1B (Gfi-1B core as above) or c-myc promoter (sequence from -868 to -840 of c-myc gene). UT-7 cells were induced to differentiate and lysates were prepared 5 days after induction of differentiation with EPO. Nuclear extracts were incubated with increasing quantities of oligonucleotides (from 1–4 μg indicated by black wedges) of wild-type or with 4 μg of mutated (indicated by a cross) oligonucleotides, precipitated with streptavidin beads and analyzed by Western blotting using antibodies against Gfi-1B or GATA-1, LSD1 and CoREST. This experiment is representative of three experiments.

    Techniques Used: Expressing, Cell Culture, Western Blot, Staining, Binding Assay, Affinity Precipitation, Labeling, Polyacrylamide Gel Electrophoresis, Sequencing, Incubation

    5) Product Images from "SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts"

    Article Title: SAFB2 enables the processing of suboptimal stem-loop structures in clustered primary miRNA transcripts

    Journal: bioRxiv

    doi: 10.1101/858647

    An unpaired region in its lower stem makes pri-miR-15a a poor substrate for the Microprocessor complex A . Proximity labeling demonstrates recruitment of DROSHA to miR-16-1, but not to miR-15a. 293T cells transfected with the HA-tagged BASU biotin ligase and constructs encoding BoxB-flanked miR-15a and miR-16 or scrambled controls thereof were incubated with biotin for 4 hrs, lysed and subjected to a streptavidin pulldown. Western blots show the expression levels of the miRNA baits (GFP), the biotin ligase (HA) and endogenous DROSHA. HA-tagged BASU becomes auto-biotinylated and serves as a positive control for the pulldown. B . A feature in its lower stem renders pri-miR-15a a poor Microprocessor substrate. MiRNA reporter assay using chimeric miR-15a/miR-26a_16-1 contructs as depicted. Reporter repression was quantified by flow cytometry 48 hrs after transduction. C . Schematic illustration of the stem-loop portion of primary miR-26a-a and miR-15a. Red circles correspond to the respective mature miRNA. Microprocessor cleavage sites are marked by black triangles. D . Pairing mutagenesis of the miR-15a lower stem enables miR-16-1-independent processing of pri-miR-15a. Ramos cells were transduced with miR-15a_16-1 cluster mutants that increased the pairing in the lower stem ( Fig. S2B ) and were analyzed for reporter repression after 48 hrs. E . A chimeric construct that combines the miR-15a lower stem with the loop and upper stem of prototypic miR-26a-2 relies on cluster assistance for efficient miRNA processing. Ramos cells expressing a control reporter or a reporter against a chimeric miR-15a/26a were transduced with the depicted constructs ( Fig. S2D ) and analysed by flow cytometry. Data are represented as mean ± SD.
    Figure Legend Snippet: An unpaired region in its lower stem makes pri-miR-15a a poor substrate for the Microprocessor complex A . Proximity labeling demonstrates recruitment of DROSHA to miR-16-1, but not to miR-15a. 293T cells transfected with the HA-tagged BASU biotin ligase and constructs encoding BoxB-flanked miR-15a and miR-16 or scrambled controls thereof were incubated with biotin for 4 hrs, lysed and subjected to a streptavidin pulldown. Western blots show the expression levels of the miRNA baits (GFP), the biotin ligase (HA) and endogenous DROSHA. HA-tagged BASU becomes auto-biotinylated and serves as a positive control for the pulldown. B . A feature in its lower stem renders pri-miR-15a a poor Microprocessor substrate. MiRNA reporter assay using chimeric miR-15a/miR-26a_16-1 contructs as depicted. Reporter repression was quantified by flow cytometry 48 hrs after transduction. C . Schematic illustration of the stem-loop portion of primary miR-26a-a and miR-15a. Red circles correspond to the respective mature miRNA. Microprocessor cleavage sites are marked by black triangles. D . Pairing mutagenesis of the miR-15a lower stem enables miR-16-1-independent processing of pri-miR-15a. Ramos cells were transduced with miR-15a_16-1 cluster mutants that increased the pairing in the lower stem ( Fig. S2B ) and were analyzed for reporter repression after 48 hrs. E . A chimeric construct that combines the miR-15a lower stem with the loop and upper stem of prototypic miR-26a-2 relies on cluster assistance for efficient miRNA processing. Ramos cells expressing a control reporter or a reporter against a chimeric miR-15a/26a were transduced with the depicted constructs ( Fig. S2D ) and analysed by flow cytometry. Data are represented as mean ± SD.

    Techniques Used: Labeling, Transfection, Construct, Incubation, Western Blot, Expressing, Positive Control, Reporter Assay, Flow Cytometry, Transduction, Mutagenesis

    6) Product Images from "Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis"

    Article Title: Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.555730

    Immunofluorescence micrograph showing the surface biotinylation of adult Clonorchis sinensis . Transformed worms were incubated with biotin and probed with streptavidin-FITC. The short-chain thiol-cleavable biotin was incorporated only into the outer surface of the tegument (A) . Streptavidin-FITC did not bind to the tegument of non-biotinylated worms (B) . Bright field showing worm biotin probed with streptavidin-FITC (C) .
    Figure Legend Snippet: Immunofluorescence micrograph showing the surface biotinylation of adult Clonorchis sinensis . Transformed worms were incubated with biotin and probed with streptavidin-FITC. The short-chain thiol-cleavable biotin was incorporated only into the outer surface of the tegument (A) . Streptavidin-FITC did not bind to the tegument of non-biotinylated worms (B) . Bright field showing worm biotin probed with streptavidin-FITC (C) .

    Techniques Used: Immunofluorescence, Transformation Assay, Incubation

    7) Product Images from "Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity"

    Article Title: Silencing of OB-RGRP in mouse hypothalamic arcuate nucleus increases leptin receptor signaling and prevents diet-induced obesity

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

    doi: 10.1073/pnas.0706671104

    OB-RGRP interacts with OB-R and regulates its cell surface expression. ( A ) HeLa cells were transduced with Ad:OB-Ra and increasing doses of Ad:OB-RGRP. Cell surface proteins were biotinylated and isolated with streptavidin-Sepharose beads. Surface (biotinylated)
    Figure Legend Snippet: OB-RGRP interacts with OB-R and regulates its cell surface expression. ( A ) HeLa cells were transduced with Ad:OB-Ra and increasing doses of Ad:OB-RGRP. Cell surface proteins were biotinylated and isolated with streptavidin-Sepharose beads. Surface (biotinylated)

    Techniques Used: Expressing, Transduction, Isolation

    8) Product Images from "The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails"

    Article Title: The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

    Journal: Developmental and comparative immunology

    doi: 10.1016/j.dci.2008.03.012

    Histone tail binding specificity of MBP-SpPHD and mutants thereof. The indicated recombinant MBP-fusion proteins were incubated with biotinylated synthetic histone tails, retrieved from the binding reaction using streptavidin beads, and bound proteins were resolved and visualized by SDS/PAGE and Western blotting with anti-MBP antibodies. One representative blot is shown for each recombinant protein tested. The MBP-tag by itself (panel e) served as a control. Lanes 1 contain 5% of the total binding reaction prior to the addition of streptavidin beads. The histone tails used are indicated above the lanes (3-6), and samples in lanes 2 (resin) did not contain any histone tails serving as negative controls.
    Figure Legend Snippet: Histone tail binding specificity of MBP-SpPHD and mutants thereof. The indicated recombinant MBP-fusion proteins were incubated with biotinylated synthetic histone tails, retrieved from the binding reaction using streptavidin beads, and bound proteins were resolved and visualized by SDS/PAGE and Western blotting with anti-MBP antibodies. One representative blot is shown for each recombinant protein tested. The MBP-tag by itself (panel e) served as a control. Lanes 1 contain 5% of the total binding reaction prior to the addition of streptavidin beads. The histone tails used are indicated above the lanes (3-6), and samples in lanes 2 (resin) did not contain any histone tails serving as negative controls.

    Techniques Used: Binding Assay, Recombinant, Incubation, SDS Page, Western Blot

    9) Product Images from "The binding landscape of a partially-selective isopeptidase inhibitor with potent pro-death activity, based on the bis(arylidene)cyclohexanone scaffold"

    Article Title: The binding landscape of a partially-selective isopeptidase inhibitor with potent pro-death activity, based on the bis(arylidene)cyclohexanone scaffold

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0259-1

    Dynamics of 2c-biotin uptake and accumulation in cells. a IMR90-E1A cells were treated for 24 h as indicated. Cell death was scored as PI positivity by cytofluorimetric analysis. 2c and 2c-biotin were used at 10 µM. NAC was 2 mM. Mean ± SD; n = 3. b IMR90-E1A cells grown on coverslips were treated for 6 h as indicated. Cells were fixed and analysed with confocal microscope using the same parameters of acquisition. 2c-biotin was revealed using streptavidin-TRITC. Quantitative analysis of the fluorescence intensity was performed using the Metamorph software. c IMR90-E1A cells were treated with 2c-biotin [2 µM] for the indicated times. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Arrows point to cytosolic accumulation of 2c-biotin. The dotted square indicates the enlarged area. Images were obtained with an epifluorescence microscopy. Bar 30 µm. d IMR90-E1A cells were treated with 2c-biotin [2 µM] for 2 h. When used in pre-treatment, NAC was 2 mM. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Images were obtained with an epifluorescence microscopy. Bar 30 µm. e IMR90-E1A cells were treated with 2c-biotin [2 µM] for 2 h. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Images were obtained with an epifluorescence microscopy. A mitotic cell is shown. Bar 50 µm. Images are shown in pseudocolors
    Figure Legend Snippet: Dynamics of 2c-biotin uptake and accumulation in cells. a IMR90-E1A cells were treated for 24 h as indicated. Cell death was scored as PI positivity by cytofluorimetric analysis. 2c and 2c-biotin were used at 10 µM. NAC was 2 mM. Mean ± SD; n = 3. b IMR90-E1A cells grown on coverslips were treated for 6 h as indicated. Cells were fixed and analysed with confocal microscope using the same parameters of acquisition. 2c-biotin was revealed using streptavidin-TRITC. Quantitative analysis of the fluorescence intensity was performed using the Metamorph software. c IMR90-E1A cells were treated with 2c-biotin [2 µM] for the indicated times. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Arrows point to cytosolic accumulation of 2c-biotin. The dotted square indicates the enlarged area. Images were obtained with an epifluorescence microscopy. Bar 30 µm. d IMR90-E1A cells were treated with 2c-biotin [2 µM] for 2 h. When used in pre-treatment, NAC was 2 mM. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Images were obtained with an epifluorescence microscopy. Bar 30 µm. e IMR90-E1A cells were treated with 2c-biotin [2 µM] for 2 h. After fixation cells were incubated with streptavidin-TRITC. Nuclei were visualized using Hoechst 33,342. Images were obtained with an epifluorescence microscopy. A mitotic cell is shown. Bar 50 µm. Images are shown in pseudocolors

    Techniques Used: Microscopy, Fluorescence, Software, Incubation, Epifluorescence Microscopy

    Identification of target isopeptidases by biotin pull-down experiments. a/b/c/d/e IMR90-E1A cells were treated for 2 h with 10 µM 2c-biotin or left untreated. 2c-botin was pre-incubated with 5 mM of NAC, as a negative control. Cellular proteins were purified by 2c-biotin pull-down using streptavidin-agarose beads. Protein complexes were separated by SDS/PAGE and immunoblotted using the indicated antibodies. Inputs are included for enrichment comparison. In the case of USP18 cells were pre-treated for 24 h with interferon-α. f IMR90-E1A cells were treated for 2 h with the indicated concentrations of 2c-biotin or left untreated. As a control, 2c-biotin was pre-incubated with 5 mM NAC. Cellular proteins were purified by 2c-biotin pull-down. Protein complexes were separated by SDS/PAGE and immunoblotted using the anti-USP14 or the anti-UCH-L5 antibodies. Inputs are included for enrichment comparison
    Figure Legend Snippet: Identification of target isopeptidases by biotin pull-down experiments. a/b/c/d/e IMR90-E1A cells were treated for 2 h with 10 µM 2c-biotin or left untreated. 2c-botin was pre-incubated with 5 mM of NAC, as a negative control. Cellular proteins were purified by 2c-biotin pull-down using streptavidin-agarose beads. Protein complexes were separated by SDS/PAGE and immunoblotted using the indicated antibodies. Inputs are included for enrichment comparison. In the case of USP18 cells were pre-treated for 24 h with interferon-α. f IMR90-E1A cells were treated for 2 h with the indicated concentrations of 2c-biotin or left untreated. As a control, 2c-biotin was pre-incubated with 5 mM NAC. Cellular proteins were purified by 2c-biotin pull-down. Protein complexes were separated by SDS/PAGE and immunoblotted using the anti-USP14 or the anti-UCH-L5 antibodies. Inputs are included for enrichment comparison

    Techniques Used: Incubation, Negative Control, Purification, SDS Page

    10) Product Images from "A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis"

    Article Title: A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis

    Journal: bioRxiv

    doi: 10.1101/2021.05.19.444771

    The function of ERGIC-ERES contact in ERGIC-COPII formation (A) HEK293T cells were transfected with control or siRNAs against TMED9. After 72 h, the cells were incubated in nutrient-rich medium or starved in EBSS for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins. Quantification shows relative SEC12 relocation to the ERGIC under the indicated conditions. The control siRNA transfection group with nutrient-rich medium treatment was set as 1.00. The blots are representative of at least three independent experiments. (B) SIM analysis of ERGIC and COPII in HeLa control cells and TMED9 KD with ERGIC-53 and SEC31A antibodies. The cells were starved in EBSS for 1 h. (C) Quantification of COPII overlap area with the ERGIC (μm 2 /100 μm 2 ERGIC area) as shown in (B). Error bars represent standard deviations of > 150 cells from three independent experiments ( > 50 cell per experiment). P-value was obtained from two-tailed t-test. (D) The structure of the SEC12 cytoplasmic domain (PDB:5tf2). The values shown in the figure represent the dimension of SEC12. Structure model was created by PyMOL. (E) HEK293T cells were infected with RUSH-SEC12 lentivirus. The cells were treated with or without 40 μM biotin for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins. Quantification shows the percentage of SEC12 relocation to the ERGIC under the indicated conditions. (F) SIM analysis of ERGIC and COPII in HeLa cells stably expressing RUSH-SEC12. The cells were transfected with siRNAs ag ainst SEC12 and TMED9, incubated in nutrient-rich medium or EBSS for 1 h with or without 40 μM biotin for 1 h, and labeled with ERGIC-53 and SEC31A antibodies. (G) Quantification of COPII overlap area with the ERGIC (μm 2 /100 μm 2 ERGIC area) as shown in (F). Error bars represent standard deviations of > 150 cells from three independent experiments ( > 50 cells per experiment). P-value was obtained from two-tailed t-test. (H) Immunoblot showing the transactivation of SAR1 on the liposome via SEC12-bound beads. Beads with or without SEC12 were incubated with liposomes with or without TMED9-CT, together with indicated variants of SAR1 in the presence or absence of GTP. After reaction, the liposomes were isolated followed by immunoblot. Quantification was based on the ratio of SAR1 with the group of highest transactivation (with SEC12, TM9-CT, SAR1, and 0.15 nM GTP set as 1.00). The blots are representative of at least three independent experiments. (I) Fluorescence imaging showing the recruitment of SAR1-BFP to the TMED9-CT-labeled liposomes attached to streptavidin agarose beads. Beads with or without SEC12 were incubated with beads coated with control liposomes or TMED9-CT. Indicated SAR1-BFP variants with GTP were incubated with the indicated combination of beads. Confocal imaging was performed to analyze the recruitment of SAR1-BFP to the liposomes on the beads in contact with beads with SEC12. (J) Quantification of SAR1 recruitment ratio as shown in (I). Error bars represent standard deviations of > 150 beads with liposomes from three independent experiments ( > 50 beads with liposomes per experiment). P-value obtained from two-tailed t-test.
    Figure Legend Snippet: The function of ERGIC-ERES contact in ERGIC-COPII formation (A) HEK293T cells were transfected with control or siRNAs against TMED9. After 72 h, the cells were incubated in nutrient-rich medium or starved in EBSS for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins. Quantification shows relative SEC12 relocation to the ERGIC under the indicated conditions. The control siRNA transfection group with nutrient-rich medium treatment was set as 1.00. The blots are representative of at least three independent experiments. (B) SIM analysis of ERGIC and COPII in HeLa control cells and TMED9 KD with ERGIC-53 and SEC31A antibodies. The cells were starved in EBSS for 1 h. (C) Quantification of COPII overlap area with the ERGIC (μm 2 /100 μm 2 ERGIC area) as shown in (B). Error bars represent standard deviations of > 150 cells from three independent experiments ( > 50 cell per experiment). P-value was obtained from two-tailed t-test. (D) The structure of the SEC12 cytoplasmic domain (PDB:5tf2). The values shown in the figure represent the dimension of SEC12. Structure model was created by PyMOL. (E) HEK293T cells were infected with RUSH-SEC12 lentivirus. The cells were treated with or without 40 μM biotin for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins. Quantification shows the percentage of SEC12 relocation to the ERGIC under the indicated conditions. (F) SIM analysis of ERGIC and COPII in HeLa cells stably expressing RUSH-SEC12. The cells were transfected with siRNAs ag ainst SEC12 and TMED9, incubated in nutrient-rich medium or EBSS for 1 h with or without 40 μM biotin for 1 h, and labeled with ERGIC-53 and SEC31A antibodies. (G) Quantification of COPII overlap area with the ERGIC (μm 2 /100 μm 2 ERGIC area) as shown in (F). Error bars represent standard deviations of > 150 cells from three independent experiments ( > 50 cells per experiment). P-value was obtained from two-tailed t-test. (H) Immunoblot showing the transactivation of SAR1 on the liposome via SEC12-bound beads. Beads with or without SEC12 were incubated with liposomes with or without TMED9-CT, together with indicated variants of SAR1 in the presence or absence of GTP. After reaction, the liposomes were isolated followed by immunoblot. Quantification was based on the ratio of SAR1 with the group of highest transactivation (with SEC12, TM9-CT, SAR1, and 0.15 nM GTP set as 1.00). The blots are representative of at least three independent experiments. (I) Fluorescence imaging showing the recruitment of SAR1-BFP to the TMED9-CT-labeled liposomes attached to streptavidin agarose beads. Beads with or without SEC12 were incubated with beads coated with control liposomes or TMED9-CT. Indicated SAR1-BFP variants with GTP were incubated with the indicated combination of beads. Confocal imaging was performed to analyze the recruitment of SAR1-BFP to the liposomes on the beads in contact with beads with SEC12. (J) Quantification of SAR1 recruitment ratio as shown in (I). Error bars represent standard deviations of > 150 beads with liposomes from three independent experiments ( > 50 beads with liposomes per experiment). P-value obtained from two-tailed t-test.

    Techniques Used: Transfection, Incubation, Two Tailed Test, Infection, Stable Transfection, Expressing, Labeling, Isolation, Fluorescence, Imaging

    11) Product Images from "Enhanced Binding of Poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 Promoter via an Extended Cytosine-Adenosine Repeat"

    Article Title: Enhanced Binding of Poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 Promoter via an Extended Cytosine-Adenosine Repeat

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008743

    Identification of proteins that bind to the (CA) 12 oligonucleotide sequence in vitro . A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA) 12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1 . The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).
    Figure Legend Snippet: Identification of proteins that bind to the (CA) 12 oligonucleotide sequence in vitro . A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA) 12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1 . The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).

    Techniques Used: Sequencing, In Vitro, Incubation, Binding Assay, SDS Page, Silver Staining, Mass Spectrometry, Molecular Weight, Western Blot, Negative Control, Agarose Gel Electrophoresis, Staining

    12) Product Images from "Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion *"

    Article Title: Proteomic Analysis Reveals a Role for RSK in p120-catenin Phosphorylation and Melanoma Cell-Cell Adhesion *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA119.001811

    Proteomic strategy to identify RSK proximity partners. A , Schematic representation of the different bait proteins analyzed using BioID. Each RSK isoforms was fused at its N-terminus with a Flag epitope followed by the mutant E. coli biotin conjugating protein BirA-R118G (BirA*) in a tetracycline inducible system. In the presence of tetracycline and biotin, the expressed baits were allowed to biotinylate proximity cellular components on lysine residues. Following cell lysis using stringent conditions, biotinylated proteins were affinity-purified using streptavidin beads. Streptavidin-bound proteins were washed and subjected to trypsin proteolysis, and the peptides were identified using LC-MS/MS. B , HEK293 Flp-In T-REx stable cell lines were treated with tetracycline in the presence of biotin for 24 h to induce bait expression and proximity biotinylation. Bait expression and associated biotinylation patterns were monitored using Flag antibodies and streptavidin-HRP, respectively. C , HEK293 Flp-In T-REx stable cell lines expressing bait proteins were imaged using immunofluorescence microscopy. Cells were stained with Flag antibodies and DAPI to visualize nuclei. Scale bars, 10 μm. D , HEK293 cells transfected with HA-tagged RSK1–4 were lysed using non-ionic detergents. Homogenate cell lysates were divided into three different fractions (H, Homogenate; S, Soluble; P, Pellet). Immunoblotting results were quantified for intensity using MultiGauge software.
    Figure Legend Snippet: Proteomic strategy to identify RSK proximity partners. A , Schematic representation of the different bait proteins analyzed using BioID. Each RSK isoforms was fused at its N-terminus with a Flag epitope followed by the mutant E. coli biotin conjugating protein BirA-R118G (BirA*) in a tetracycline inducible system. In the presence of tetracycline and biotin, the expressed baits were allowed to biotinylate proximity cellular components on lysine residues. Following cell lysis using stringent conditions, biotinylated proteins were affinity-purified using streptavidin beads. Streptavidin-bound proteins were washed and subjected to trypsin proteolysis, and the peptides were identified using LC-MS/MS. B , HEK293 Flp-In T-REx stable cell lines were treated with tetracycline in the presence of biotin for 24 h to induce bait expression and proximity biotinylation. Bait expression and associated biotinylation patterns were monitored using Flag antibodies and streptavidin-HRP, respectively. C , HEK293 Flp-In T-REx stable cell lines expressing bait proteins were imaged using immunofluorescence microscopy. Cells were stained with Flag antibodies and DAPI to visualize nuclei. Scale bars, 10 μm. D , HEK293 cells transfected with HA-tagged RSK1–4 were lysed using non-ionic detergents. Homogenate cell lysates were divided into three different fractions (H, Homogenate; S, Soluble; P, Pellet). Immunoblotting results were quantified for intensity using MultiGauge software.

    Techniques Used: FLAG-tag, Mutagenesis, Lysis, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing, Immunofluorescence, Microscopy, Staining, Transfection, Software

    p120ctn proximity partners are regulated by Ser320 phosphorylation. A , HEK293 Flp-In T-REx stable cell lines were treated with tetracycline in the presence of biotin for 24 h to induce bait expression and proximity biotinylation. Bait expression and biotinylation patterns were monitored using a Flag antibody and Streptavidin-HRP, respectively. B , Venn diagram showing the overlap of proximity interactors identified by BioID with the three different baits. Only the preys with a SAINT Score > 0.5 were considered. C , Dotplot of selected interaction partners identified by BioID. The node color represents the average spectral count sum ( n = 3), the node edge color corresponds to the SAINT Score and the node size displays the relative abundance of a given prey across the three conditions compared. D , A375 cells were stained for endogenous p120ctn (green) and 4E-T (red), and pictures were taken with a confocal microscope. White dots represent p120ctn and 4E-T colocalization. E , Graph representing the relative fluorescence intensity of 4E-T and p120ctn along the area shown by the red arrow in ( D ). F , Scatter plot representing the correlation between in p120ctn and 4E-T staining. The red line represents the Pearson correlation trendline. Scale bars, 10 μm.
    Figure Legend Snippet: p120ctn proximity partners are regulated by Ser320 phosphorylation. A , HEK293 Flp-In T-REx stable cell lines were treated with tetracycline in the presence of biotin for 24 h to induce bait expression and proximity biotinylation. Bait expression and biotinylation patterns were monitored using a Flag antibody and Streptavidin-HRP, respectively. B , Venn diagram showing the overlap of proximity interactors identified by BioID with the three different baits. Only the preys with a SAINT Score > 0.5 were considered. C , Dotplot of selected interaction partners identified by BioID. The node color represents the average spectral count sum ( n = 3), the node edge color corresponds to the SAINT Score and the node size displays the relative abundance of a given prey across the three conditions compared. D , A375 cells were stained for endogenous p120ctn (green) and 4E-T (red), and pictures were taken with a confocal microscope. White dots represent p120ctn and 4E-T colocalization. E , Graph representing the relative fluorescence intensity of 4E-T and p120ctn along the area shown by the red arrow in ( D ). F , Scatter plot representing the correlation between in p120ctn and 4E-T staining. The red line represents the Pearson correlation trendline. Scale bars, 10 μm.

    Techniques Used: Stable Transfection, Expressing, Staining, Microscopy, Fluorescence

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    Article Snippet: .. Biotin incorporation was assessed by incubating blots with streptavidin-HRP (1:1000 v /v , GE Healthcare) and vimentin levels estimated by western blot with anti-vimentin V9 monoclonal antibody (1:1000 v /v; sc-6260, Santa Cruz), followed by enhanced chemiluminescent (ECL) detection (GE Healthcare). ..

    Article Title: Performance of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies in South Africa
    Article Snippet: .. Expression yields and in vivo biotinylation of the N domains and S ectodomain were assessed by western blot using a streptavidin-HRP conjugate probe (GE Healthcare, Chicago, Illinois, United states of America). ..

    Incubation:

    Article Title: ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A
    Article Snippet: .. Each array antibody membrane was blocked, then incubated with protein samples (0.5 mg/mL) overnight at 4 °C and continuing incubated with HRP linked Streptavidin conjugate for 1 h. The spots were visualized by enhanced chemiluminescence (ECL) (Amersham, Chicago, IL, USA) and the signal densities were analyzed with ImageJ software (National Institute of Health, Bethesda, MD, USA). ..

    Article Title: CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein
    Article Snippet: .. To reveal biotinylated proteins, the membrane was blocked with BSA blocking buffer (PBS, 1% BSA, 0.2% Triton X-100) and incubated 1 hour at RT or 16 hours at 4°C with HRP-coupled streptavidin (RPN1231VS, GE Healthcare) diluted 1:20000 in BSA blocking buffer. ..

    Software:

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    Expressing:

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    In Vivo:

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    GE Healthcare streptavidin sepharose beads
    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 <t>streptavidin-Sepharose</t> 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.
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    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

    DOX-PCB nanoparticle surface binding assay. The inherently fluorescent DOX-PCB NPs were incubated with streptavidin-coated Sepharose microbeads and uncoated Sepharose control microbeads. ( A ) The fluorescent DOX-PCB NPs bind to the streptavidin-coated beads outlining their surface. This indicates the presence of the biotin-terminated PEG tail on the surface of the nanoparticles. ( B ) No binding was seen with the uncoated Sepharose control beads.

    Journal: Pharmaceutical research

    Article Title: Drug Delivery Nanoparticles with Locally Tunable Toxicity Made Entirely from a Light-Activatable Prodrug of Doxorubicin

    doi: 10.1007/s11095-017-2205-4

    Figure Lengend Snippet: DOX-PCB nanoparticle surface binding assay. The inherently fluorescent DOX-PCB NPs were incubated with streptavidin-coated Sepharose microbeads and uncoated Sepharose control microbeads. ( A ) The fluorescent DOX-PCB NPs bind to the streptavidin-coated beads outlining their surface. This indicates the presence of the biotin-terminated PEG tail on the surface of the nanoparticles. ( B ) No binding was seen with the uncoated Sepharose control beads.

    Article Snippet: To replace the beads’ 20% ethanol storage solutions with water, samples of 50 µl of Streptavidin Sepharose High Performance beads (GE Healthcare Life Sciences, Little Chalfont, UK) and uncoated Sepharose control beads (Sepharose CL-4B, GE Healthcare Life Sciences, Little Chalfont, UK) were each diluted by adding 1 ml Milli-Q water, then spun down with subsequent removal of 1 ml supernatant, followed by the addition of 50 µl fresh Milli-Q water.

    Techniques: Binding Assay, Incubation

    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

    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