flag peptide  (Millipore)


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    Custom Peptides
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    PEPscreen Custom Peptide LIbraries Suitable for robotic production of peptide microarrays Screen mulitple proteotyptic peptides and rapidly characterize and select mass spec compatible peptides Get results quick with delivery of your peptide library in less than 7 business days Sequence Length 6 to 20 amino acids Amount 0 5 2 mg or 2 5 mg Modifications Comprehensive offering including phosphorylation biotin fluorescein stable isotopes etc Format Supplied dry in a 96 well tube rack Options Normalization and aliquoting stock and copy libraries Minimum library size of 24 peptidesRequest more informationUse our free design tool to design your libraryAQUA Peptides Accurately quantitate low abundance proteins Measure site specific phosphorylation states Validate gene silencing at the protein level Custom peptides are available either without stable isotope labeling or with stable isotope labeling Stable isotope labeled AQUA TM Peptide seqeunces contain one isotopically labeled amino acid Sequence Length 5 to 30 amino acids Amount 5 x 1 nmole Purity 95 by RP HPLC Modifications phosphorylation Ser Thr Tyr carboxymethylated Cys carbamidomethylated Cys hydroxyproline N terminal biotin please inquire regarding other modifications Quantification by amino acid analysis AAA Format Supplied dryRequest more information
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    Structured Review

    Millipore flag peptide
    Custom Peptides
    PEPscreen Custom Peptide LIbraries Suitable for robotic production of peptide microarrays Screen mulitple proteotyptic peptides and rapidly characterize and select mass spec compatible peptides Get results quick with delivery of your peptide library in less than 7 business days Sequence Length 6 to 20 amino acids Amount 0 5 2 mg or 2 5 mg Modifications Comprehensive offering including phosphorylation biotin fluorescein stable isotopes etc Format Supplied dry in a 96 well tube rack Options Normalization and aliquoting stock and copy libraries Minimum library size of 24 peptidesRequest more informationUse our free design tool to design your libraryAQUA Peptides Accurately quantitate low abundance proteins Measure site specific phosphorylation states Validate gene silencing at the protein level Custom peptides are available either without stable isotope labeling or with stable isotope labeling Stable isotope labeled AQUA TM Peptide seqeunces contain one isotopically labeled amino acid Sequence Length 5 to 30 amino acids Amount 5 x 1 nmole Purity 95 by RP HPLC Modifications phosphorylation Ser Thr Tyr carboxymethylated Cys carbamidomethylated Cys hydroxyproline N terminal biotin please inquire regarding other modifications Quantification by amino acid analysis AAA Format Supplied dryRequest more information
    https://www.bioz.com/result/flag peptide/product/Millipore
    Average 99 stars, based on 336 article reviews
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    flag peptide - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage"

    Article Title: The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36235-0

    UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E , F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G , H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) .
    Figure Legend Snippet: UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E , F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G , H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) .

    Techniques Used: Non-Homologous End Joining, Reporter Assay, Transfection, Plasmid Preparation, Clonogenic Cell Survival Assay, Stable Transfection, Expressing, Mutagenesis

    UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability. (A) U2OS cells transfected with a plasmid expressing FLAG-UCHL3 were treated with phleomycin. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (B) U2OS cells transfected with a plasmid expressing either FLAG-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) were treated with phleomycin or mock treated. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (C) U2OS cells transfected with a plasmid expressing either FLAG or FLAG-UCHL3 were treated with phleomycin for 1 hour and further cultured for the indicated time periods after removal of phleomycin. Cells were processed for immunoprecipitation with anti-FLAG antibody followed by immunoblotting analyses with the indicated antibodies. (D) U2OS cells transfected with the indicated siRNAs were further transfected with a plasmid expressing either GFP-UCHL3 or GFP. Following phleomycin treatment, cell extracts were subjected to immunoprecipitation with an anti-GFP antibody and ensuing immunoblotting analysis with the indicated antibodies. The plasmid expressing GFP was used as a negative control. (E) .
    Figure Legend Snippet: UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability. (A) U2OS cells transfected with a plasmid expressing FLAG-UCHL3 were treated with phleomycin. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (B) U2OS cells transfected with a plasmid expressing either FLAG-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) were treated with phleomycin or mock treated. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (C) U2OS cells transfected with a plasmid expressing either FLAG or FLAG-UCHL3 were treated with phleomycin for 1 hour and further cultured for the indicated time periods after removal of phleomycin. Cells were processed for immunoprecipitation with anti-FLAG antibody followed by immunoblotting analyses with the indicated antibodies. (D) U2OS cells transfected with the indicated siRNAs were further transfected with a plasmid expressing either GFP-UCHL3 or GFP. Following phleomycin treatment, cell extracts were subjected to immunoprecipitation with an anti-GFP antibody and ensuing immunoblotting analysis with the indicated antibodies. The plasmid expressing GFP was used as a negative control. (E) .

    Techniques Used: Activity Assay, Non-Homologous End Joining, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Negative Control, Mutagenesis, Cell Culture

    2) Product Images from "A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment *"

    Article Title: A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment *

    Journal:

    doi: 10.1074/jbc.M806008200

    The SECIS element promotes formation of a stable SID-RBD complex. Wild-type or penta-alanine mutant recombinant XH-SID ( SID ) was incubated with equimolar amounts of FLAG-RBD and 32 P-trace-labeled wild-type ( wt ) or mutant ( mt ) SECIS elements. The proteins
    Figure Legend Snippet: The SECIS element promotes formation of a stable SID-RBD complex. Wild-type or penta-alanine mutant recombinant XH-SID ( SID ) was incubated with equimolar amounts of FLAG-RBD and 32 P-trace-labeled wild-type ( wt ) or mutant ( mt ) SECIS elements. The proteins

    Techniques Used: Mutagenesis, Recombinant, Incubation, Labeling

    3) Product Images from "The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity"

    Article Title: The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00324-13

    Copurification of CydX with CydA. (A) Purification of CydA-His and CydX-SPA on a Ni-NTA column when CydA-His is expressed compared to purification of CydX-SPA on a Ni-NTA column in the absence of CydA-His. (B) Purification of CydA-His and AcrZ-SPA on a Ni-NTA column when CydA-His is expressed compared to purification of AcrZ-SPA on a Ni-NTA column in the absence of CydA-His. Strains were grown under aerobic conditions at 30°C. Cell lysates were run through a Ni-NTA column to purify CydA-His, and samples of these purified proteins were tested for CydX or AcrZ small-protein copurification by probing Western blots with an alkaline phosphatase-conjugated anti-FLAG (α-FLAG) monoclonal antibody. CydA-His purification was confirmed by probing identical Western blots of the purified protein samples with a horseradish peroxidase (HRP)-conjugated anti-His (α-His) monoclonal antibody. Antibody signal for the anti-FLAG Western blots was visualized using the Lumiphos substrate, and antibody signal was visualized for the anti-6×His Western blots using the SuperSignal West Femto substrate. The sample abbreviations are as follows: CL, cleared lysate; FT, flowthrough; W1, wash 1; W2, wash 2; W3, wash 3; E, eluate.
    Figure Legend Snippet: Copurification of CydX with CydA. (A) Purification of CydA-His and CydX-SPA on a Ni-NTA column when CydA-His is expressed compared to purification of CydX-SPA on a Ni-NTA column in the absence of CydA-His. (B) Purification of CydA-His and AcrZ-SPA on a Ni-NTA column when CydA-His is expressed compared to purification of AcrZ-SPA on a Ni-NTA column in the absence of CydA-His. Strains were grown under aerobic conditions at 30°C. Cell lysates were run through a Ni-NTA column to purify CydA-His, and samples of these purified proteins were tested for CydX or AcrZ small-protein copurification by probing Western blots with an alkaline phosphatase-conjugated anti-FLAG (α-FLAG) monoclonal antibody. CydA-His purification was confirmed by probing identical Western blots of the purified protein samples with a horseradish peroxidase (HRP)-conjugated anti-His (α-His) monoclonal antibody. Antibody signal for the anti-FLAG Western blots was visualized using the Lumiphos substrate, and antibody signal was visualized for the anti-6×His Western blots using the SuperSignal West Femto substrate. The sample abbreviations are as follows: CL, cleared lysate; FT, flowthrough; W1, wash 1; W2, wash 2; W3, wash 3; E, eluate.

    Techniques Used: Copurification, Purification, Western Blot

    Copurification of oxidase activity with CydX. TMPD oxidase activity of extracts isolated from E. coli MG1655 cells and cells expressing CydX-SPA and AcrZ-SPA and purified on an anti-FLAG column. Oxidase activity was determined by measuring the oxidation of N , N , N ′, N ′-tetramethyl- p -phenylenediamine by the purification eluates incubated at room temperature. TMPD oxidation was measured by assaying absorbance at 611 nm using a NanoDrop spectrophotometer. Since TMPD is oxidized by air, background oxidation was measured in a blank sample in which no protein sample was added. Relative levels of activity based on absorbance readings at 611 nm are reported. (Inset) Relative levels of purified SPA-tagged protein from the three purified samples used in the TMPD assay. The Western blot of purification samples was probed with anti-FLAG antibody and visualized using the Lumiphos substrate.
    Figure Legend Snippet: Copurification of oxidase activity with CydX. TMPD oxidase activity of extracts isolated from E. coli MG1655 cells and cells expressing CydX-SPA and AcrZ-SPA and purified on an anti-FLAG column. Oxidase activity was determined by measuring the oxidation of N , N , N ′, N ′-tetramethyl- p -phenylenediamine by the purification eluates incubated at room temperature. TMPD oxidation was measured by assaying absorbance at 611 nm using a NanoDrop spectrophotometer. Since TMPD is oxidized by air, background oxidation was measured in a blank sample in which no protein sample was added. Relative levels of activity based on absorbance readings at 611 nm are reported. (Inset) Relative levels of purified SPA-tagged protein from the three purified samples used in the TMPD assay. The Western blot of purification samples was probed with anti-FLAG antibody and visualized using the Lumiphos substrate.

    Techniques Used: Copurification, Activity Assay, Isolation, Expressing, Purification, Incubation, Spectrophotometry, Western Blot

    Reciprocal copurification of CydA with CydX. Purification of CydX-SPA and CydA-His on an anti-FLAG column followed by purification on a calmodulin binding protein (CBP) column. For a control, extracts from a CydA-His strain lacking CydX-SPA were purified and analyzed for the presence of CydA-His. Strains were grown under low-oxygen conditions at 37°C. Cells were harvested by centrifugation and lysed using a cell breaker. Lysates were run through a column containing anti-3×FLAG-bound resin to purify the CydX-SPA, and samples of these purified proteins were tested for CydA-His copurification by probing Western blots with a HRP-conjugated anti-His (α-His) monoclonal antibody. CydX-SPA purification was confirmed by probing identical Western blots of the purification samples with an alkaline phosphatase-conjugated anti-FLAG (α-FLAG) monoclonal antibody. Antibody signal was visualized for the anti-FLAG Western blots using the Lumiphos substrate, and antibody signal was visualized for the anti-6×His Western blots using the SuperSignal West Femto substrate. The sample abbreviations are as follows: CL, cleared lysate; FT, flowthrough; W, wash; E, eluate.
    Figure Legend Snippet: Reciprocal copurification of CydA with CydX. Purification of CydX-SPA and CydA-His on an anti-FLAG column followed by purification on a calmodulin binding protein (CBP) column. For a control, extracts from a CydA-His strain lacking CydX-SPA were purified and analyzed for the presence of CydA-His. Strains were grown under low-oxygen conditions at 37°C. Cells were harvested by centrifugation and lysed using a cell breaker. Lysates were run through a column containing anti-3×FLAG-bound resin to purify the CydX-SPA, and samples of these purified proteins were tested for CydA-His copurification by probing Western blots with a HRP-conjugated anti-His (α-His) monoclonal antibody. CydX-SPA purification was confirmed by probing identical Western blots of the purification samples with an alkaline phosphatase-conjugated anti-FLAG (α-FLAG) monoclonal antibody. Antibody signal was visualized for the anti-FLAG Western blots using the Lumiphos substrate, and antibody signal was visualized for the anti-6×His Western blots using the SuperSignal West Femto substrate. The sample abbreviations are as follows: CL, cleared lysate; FT, flowthrough; W, wash; E, eluate.

    Techniques Used: Copurification, Purification, Binding Assay, Centrifugation, Western Blot

    4) Product Images from "Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS"

    Article Title: Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS

    Journal: Cell Research

    doi: 10.1038/s41422-018-0107-6

    OTUD4 deubiquitinates and stabilizes MAVS. a Denature-immunoprecipitation (Denature-IP) (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA or anti-OTUD4) of HEK293 cells transfected with plasmids encoding FLAG-MAVS, HA-Ubiquitin and empty vector, OTUD4, or OTUD4(C45A) for 24 h. b In vitro deubiquitination analysis of ubiquitin-modified MAVS eluted from anti-FLAG precipitates by FLAG peptide of HEK293 cells transfected with FLAG-MAVS and HA-ubiquitin incubated with in vitro generated OTUD4 or OTUD4 C45A obtained from an in vitro transcription and translation kit. c Denature-IP (with anti-MAVS) and immunoblot analysis (with anti-K48 linkage polyubiquitin, anti-MAVS, anti-OTUD4 or anti-Tubulin) of Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl MLFs treated with 4-OH Tam (1 μM) for 3 days followed by MG132 treatment for 2 h prior to VSV infection for 0–6 h. d Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl MLFs treated with 4-OH Tam infected with VSV for 0–12 h in the presence or absence of cycloheximide (100 μg/ml). The relative intensities of MAVS were determined by normalizing the intensities of MAVS by the respective intensities of GAPDH. e Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl BMDMs treated with 4-OH Tam followed by infection with VSV for 8 h in the presence of MG132 or Baf A1 for 4 h. f Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/fl BMDMs treated with 4-OH Tam and reconstituted with Vec, OTUD4 or OTUD4(C45A) followed by infection with VSV for 0–8 h. The relative intensities of MAVS were determined by normalizing the intensities of MAVS by the respective intensities of GAPDH. g qRT-PCR analysis of Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl BMDCs treated with 4-OH Tam and reconstituted with Vec, or MAVS followed by infection with SeV for 0–6 h. h Immunoblot analysis of total and phosphorylated (p-)TBK1, IκBα, IRF3, FLAG-VISA, OTUD4 or β-Actin in cells obtained in ( g ) infected with SeV for 0–6 h. i Flow cytometry analysis (left) and microscopy imaging (right images) of the replication of GFP-VSV in cells obtained in ( g ) infected with VSV-GFP (MOI = 1). Data are representative of two or three independent experiments
    Figure Legend Snippet: OTUD4 deubiquitinates and stabilizes MAVS. a Denature-immunoprecipitation (Denature-IP) (with anti-FLAG) and immunoblot analysis (with anti-FLAG, anti-HA or anti-OTUD4) of HEK293 cells transfected with plasmids encoding FLAG-MAVS, HA-Ubiquitin and empty vector, OTUD4, or OTUD4(C45A) for 24 h. b In vitro deubiquitination analysis of ubiquitin-modified MAVS eluted from anti-FLAG precipitates by FLAG peptide of HEK293 cells transfected with FLAG-MAVS and HA-ubiquitin incubated with in vitro generated OTUD4 or OTUD4 C45A obtained from an in vitro transcription and translation kit. c Denature-IP (with anti-MAVS) and immunoblot analysis (with anti-K48 linkage polyubiquitin, anti-MAVS, anti-OTUD4 or anti-Tubulin) of Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl MLFs treated with 4-OH Tam (1 μM) for 3 days followed by MG132 treatment for 2 h prior to VSV infection for 0–6 h. d Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl MLFs treated with 4-OH Tam infected with VSV for 0–12 h in the presence or absence of cycloheximide (100 μg/ml). The relative intensities of MAVS were determined by normalizing the intensities of MAVS by the respective intensities of GAPDH. e Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl BMDMs treated with 4-OH Tam followed by infection with VSV for 8 h in the presence of MG132 or Baf A1 for 4 h. f Immunoblot analysis of MAVS, OTUD4 and GAPDH in Cre-ER Otud4 fl/fl BMDMs treated with 4-OH Tam and reconstituted with Vec, OTUD4 or OTUD4(C45A) followed by infection with VSV for 0–8 h. The relative intensities of MAVS were determined by normalizing the intensities of MAVS by the respective intensities of GAPDH. g qRT-PCR analysis of Cre-ER Otud4 fl/+ and Cre-ER Otud4 fl/fl BMDCs treated with 4-OH Tam and reconstituted with Vec, or MAVS followed by infection with SeV for 0–6 h. h Immunoblot analysis of total and phosphorylated (p-)TBK1, IκBα, IRF3, FLAG-VISA, OTUD4 or β-Actin in cells obtained in ( g ) infected with SeV for 0–6 h. i Flow cytometry analysis (left) and microscopy imaging (right images) of the replication of GFP-VSV in cells obtained in ( g ) infected with VSV-GFP (MOI = 1). Data are representative of two or three independent experiments

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, In Vitro, Modification, Incubation, Generated, Infection, Quantitative RT-PCR, Flow Cytometry, Cytometry, Microscopy, Imaging

    Identification of OTUD4 as a MAVS-interacting DUB. a Immunoprecipitation (IP, with anti-FLAG) and immunoblot (IB, with anti-FLAG and anti-HA and goat anti-mouse IgG(H+L) antibody) analysis of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged DUBs for 24 h. Cell lysates were analyzed by immunoblot with anti-FLAG or anti-HA. b Immunoprecipitation (with control IgG or anti-OTUD4) and immunoblot (with anti-OTUD4 and anti-MAVS and goat anti-mouse IgG F(ab’)2 fragment specific antibody) of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with SeV or VSV for 5–10 h. Cell lysates were analyzed by immunoblot with antibodies against the indicated proteins. The graphs show relative intensities of OTUD4 obtained by normalizing the intensities of OTUD4 to the intensities of β-Actin. c , d Immunoblot of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged OTUD4 or mutants ( c ) or with plasmids encoding GFP-OTUD4 and FLAG-tagged MAVS or truncates ( d ), lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-FLAG-HRP, anti-GFP or anti-HA. Data are representative of three ( a ) or two ( b–d ) independent experiments (mean ± S.D. in b )
    Figure Legend Snippet: Identification of OTUD4 as a MAVS-interacting DUB. a Immunoprecipitation (IP, with anti-FLAG) and immunoblot (IB, with anti-FLAG and anti-HA and goat anti-mouse IgG(H+L) antibody) analysis of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged DUBs for 24 h. Cell lysates were analyzed by immunoblot with anti-FLAG or anti-HA. b Immunoprecipitation (with control IgG or anti-OTUD4) and immunoblot (with anti-OTUD4 and anti-MAVS and goat anti-mouse IgG F(ab’)2 fragment specific antibody) of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with SeV or VSV for 5–10 h. Cell lysates were analyzed by immunoblot with antibodies against the indicated proteins. The graphs show relative intensities of OTUD4 obtained by normalizing the intensities of OTUD4 to the intensities of β-Actin. c , d Immunoblot of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged OTUD4 or mutants ( c ) or with plasmids encoding GFP-OTUD4 and FLAG-tagged MAVS or truncates ( d ), lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-FLAG-HRP, anti-GFP or anti-HA. Data are representative of three ( a ) or two ( b–d ) independent experiments (mean ± S.D. in b )

    Techniques Used: Immunoprecipitation, Transfection, Infection

    5) Product Images from "Dimerization of sortilin regulates its trafficking to extracellular vesicles"

    Article Title: Dimerization of sortilin regulates its trafficking to extracellular vesicles

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000732

    Sortilin forms homodimers on the cell surface of HEK293 cells. A , schematic of FLAG-sortilin and His 6 -sortilin. FLAG tag and His 6 tag were inserted following propeptide and 3 amino acids (Ser 78 -Ala 79 -Pro 80 ) in sortilin. SP , signal peptide; PP , propeptide. B , overexpression of FLAG-sortilin and His 6 -sortilin in HEK293 cells was validated by Western blotting. C and D , detection of binding of FLAG-sortilin and His 6 -sortilin on the cell surface of HEK293 in TR-FRET assay ( C ) and HTRF assay ( D ). Change of FRET signal by expression of His 6 -sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p
    Figure Legend Snippet: Sortilin forms homodimers on the cell surface of HEK293 cells. A , schematic of FLAG-sortilin and His 6 -sortilin. FLAG tag and His 6 tag were inserted following propeptide and 3 amino acids (Ser 78 -Ala 79 -Pro 80 ) in sortilin. SP , signal peptide; PP , propeptide. B , overexpression of FLAG-sortilin and His 6 -sortilin in HEK293 cells was validated by Western blotting. C and D , detection of binding of FLAG-sortilin and His 6 -sortilin on the cell surface of HEK293 in TR-FRET assay ( C ) and HTRF assay ( D ). Change of FRET signal by expression of His 6 -sortilin is indicated by percent change (mean ± S.D., three independent experiments). Error bars represent S.D. *, p

    Techniques Used: FLAG-tag, Over Expression, Western Blot, Binding Assay, HTRF Assay, Expressing

    Substituting the transmembrane domain of sortilin with the corresponding domain of CD43 does not decrease the dimeric form of sortilin. A , schematic of FLAG-sortilin wildtype (WT) and FLAG-sortilin CD43-TMD. The transmembrane domain of sortilin was replaced with that of CD43. SP , signal peptide; PP , propeptide. B , FLAG-sortilin WT and FLAG-sortilin CD43-TMD were transiently overexpressed in HEK293 cells, and non-reducing Western blotting was carried out using cell lysate with anti-FLAG antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. C and D , His 6 -sortilin WT or His 6 -sortilin CD43-TMD was transiently overexpressed in HEK293 cells stably overexpressing FLAG-sortilin, and immunoprecipitation was performed using anti-FLAG M2 antibody. Western blotting was carried out using whole-cell lysates ( C ) and immunoprecipitants ( D ). His 6 -sortilin CD43-TMD coprecipitated with FLAG-sortilin as well as His 6 -sortilin WT. Arrows , sortilin wildtype or sortilin CD43-TMD ( n = 3). E , in FLAG-sortilin HEK293 cells or HEK293 cells, His 6 -sortilin CD43-TMD was overexpressed. The cells were subjected to TR-FRET assay. Change of FRET signal by expression of His 6 -sortilin WT or CD43-TMD is indicated by percent change (mean ± S.D., n = 4, one independent experiment). Error bars represent S.D. *, p
    Figure Legend Snippet: Substituting the transmembrane domain of sortilin with the corresponding domain of CD43 does not decrease the dimeric form of sortilin. A , schematic of FLAG-sortilin wildtype (WT) and FLAG-sortilin CD43-TMD. The transmembrane domain of sortilin was replaced with that of CD43. SP , signal peptide; PP , propeptide. B , FLAG-sortilin WT and FLAG-sortilin CD43-TMD were transiently overexpressed in HEK293 cells, and non-reducing Western blotting was carried out using cell lysate with anti-FLAG antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. C and D , His 6 -sortilin WT or His 6 -sortilin CD43-TMD was transiently overexpressed in HEK293 cells stably overexpressing FLAG-sortilin, and immunoprecipitation was performed using anti-FLAG M2 antibody. Western blotting was carried out using whole-cell lysates ( C ) and immunoprecipitants ( D ). His 6 -sortilin CD43-TMD coprecipitated with FLAG-sortilin as well as His 6 -sortilin WT. Arrows , sortilin wildtype or sortilin CD43-TMD ( n = 3). E , in FLAG-sortilin HEK293 cells or HEK293 cells, His 6 -sortilin CD43-TMD was overexpressed. The cells were subjected to TR-FRET assay. Change of FRET signal by expression of His 6 -sortilin WT or CD43-TMD is indicated by percent change (mean ± S.D., n = 4, one independent experiment). Error bars represent S.D. *, p

    Techniques Used: Western Blot, Stable Transfection, Immunoprecipitation, Expressing

    Mutation of Cys 783 abolishes dimerization of sortilin. A , schematic of His 6 -sortilin 10CC+TMD, FLAG-sortilin WT and C783A, and His 6 -sortilin ICD+TMD WT and C783A. Cysteine 783 was replaced by alanine. SP , signal peptide; PP , propeptide. B , expression vector of His 6 -sortilin 10CC+TMD was transfected in HEK293 cells. Dimerization of His 6 -sortilin 10CC+TMD was detected in non-reducing Western blotting with anti-His 6 antibody ( n = 3). C , sortilin ICD+TMD C783A did not form homodimers in HEK293 cells in the non-reducing Western blotting ( n = 3). D and E , C783A decreased sortilin homodimers of low molecular weight in the cells ( D ) and extracellular vesicles ( E ) of HEK293 cells in non-reducing Western blotting ( n = 3). F and G , 24-h incubation with 2-FPA, an inhibitor of palmitoylation, increased sortilin homodimers of low molecular weight in HEK293 cells stably overexpressing FLAG-sortilin ( F ) and their extracellular vesicles ( G ) ( n = 3). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. IB , immunoblotting.
    Figure Legend Snippet: Mutation of Cys 783 abolishes dimerization of sortilin. A , schematic of His 6 -sortilin 10CC+TMD, FLAG-sortilin WT and C783A, and His 6 -sortilin ICD+TMD WT and C783A. Cysteine 783 was replaced by alanine. SP , signal peptide; PP , propeptide. B , expression vector of His 6 -sortilin 10CC+TMD was transfected in HEK293 cells. Dimerization of His 6 -sortilin 10CC+TMD was detected in non-reducing Western blotting with anti-His 6 antibody ( n = 3). C , sortilin ICD+TMD C783A did not form homodimers in HEK293 cells in the non-reducing Western blotting ( n = 3). D and E , C783A decreased sortilin homodimers of low molecular weight in the cells ( D ) and extracellular vesicles ( E ) of HEK293 cells in non-reducing Western blotting ( n = 3). F and G , 24-h incubation with 2-FPA, an inhibitor of palmitoylation, increased sortilin homodimers of low molecular weight in HEK293 cells stably overexpressing FLAG-sortilin ( F ) and their extracellular vesicles ( G ) ( n = 3). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. IB , immunoblotting.

    Techniques Used: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Western Blot, Molecular Weight, Incubation, Stable Transfection

    The transmembrane domain of sortilin forms homodimers via noncovalent interaction. A–D , His 6 -sortilin Full, ECD+TMD, and ICD+TMD were transiently overexpressed in HEK293 cells with stably overexpressed FLAG-sortilin Full ( A and B ) and ECD+TMD ( C and D ), respectively. Immunoprecipitation with anti-FLAG M2 antibody was performed using the cell lysates. Western blotting was carried out using whole-cell lysates ( A and C ) and immunoprecipitants ( B and D ). His 6 -sortilin Full, ECD+TMD, and ICD+TMD were coprecipitated with FLAG-sortilin Full or ECD+TMD ( B and D ) ( n = 3). IB , immunoblotting.
    Figure Legend Snippet: The transmembrane domain of sortilin forms homodimers via noncovalent interaction. A–D , His 6 -sortilin Full, ECD+TMD, and ICD+TMD were transiently overexpressed in HEK293 cells with stably overexpressed FLAG-sortilin Full ( A and B ) and ECD+TMD ( C and D ), respectively. Immunoprecipitation with anti-FLAG M2 antibody was performed using the cell lysates. Western blotting was carried out using whole-cell lysates ( A and C ) and immunoprecipitants ( B and D ). His 6 -sortilin Full, ECD+TMD, and ICD+TMD were coprecipitated with FLAG-sortilin Full or ECD+TMD ( B and D ) ( n = 3). IB , immunoblotting.

    Techniques Used: Stable Transfection, Immunoprecipitation, Western Blot

    Sortilin forms homodimers in the extracellular and intracellular domains with intermolecular disulfide bonds in HEK293 cells. A , schematic of FLAG-sortilin Full, ECD+TMD, and ICD+TMD. SP , signal peptide; PP , propeptide. B and C , protein expression of FLAG-sortilin Full, ECD+TMD, and ICD+TMD was validated in reducing ( B ) and non-reducing ( C ) Western blotting using anti-FLAG antibody. FLAG-sortilin Full and ECD+TMD form homodimers and multimers. Empty vector was used as a control. D , HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, and the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full and ECD+TMD ( n = 3). E , HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO ( Control ), 20 μmol/liter MG-132 ( MG ) or 10 μmol/liter chloroquine ( Chlo ) for 7 h, and then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not ( n = 3). F , FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- and concentration-dependent manner ( n = 3). G , following 16-h incubation of HEK293 cells ( Control ) or FLAG-sortilin ICD+TMD HEK293 cells ( ICD + TMD ) with MG-132 (5 μmol/liter) and immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. IB , immunoblotting.
    Figure Legend Snippet: Sortilin forms homodimers in the extracellular and intracellular domains with intermolecular disulfide bonds in HEK293 cells. A , schematic of FLAG-sortilin Full, ECD+TMD, and ICD+TMD. SP , signal peptide; PP , propeptide. B and C , protein expression of FLAG-sortilin Full, ECD+TMD, and ICD+TMD was validated in reducing ( B ) and non-reducing ( C ) Western blotting using anti-FLAG antibody. FLAG-sortilin Full and ECD+TMD form homodimers and multimers. Empty vector was used as a control. D , HEK293 cells transiently overexpressing FLAG-sortilin Full or ECD+TMD were treated with a cross-linker, BS3, and the cell lysates were used for reducing Western blotting with anti-FLAG antibody, showing dimerization of sortilin Full and ECD+TMD ( n = 3). E , HEK293 cells stably overexpressing FLAG-sortilin ICD+TMD (FLAG-sortilin ICD+TMD HEK293 cells) were incubated with DMSO ( Control ), 20 μmol/liter MG-132 ( MG ) or 10 μmol/liter chloroquine ( Chlo ) for 7 h, and then reducing Western blotting was performed using anti-sortilin antibody. MG-132 increased the protein expression of FLAG-sortilin ICD+TMD, but chloroquine did not ( n = 3). F , FLAG-sortilin ICD+TMD HEK293 cells were incubated with DMSO or MG-132 (2–20 μmol/liter) for 7 or 24 h. MG-132 increased FLAG-sortilin ICD+TMD in a time- and concentration-dependent manner ( n = 3). G , following 16-h incubation of HEK293 cells ( Control ) or FLAG-sortilin ICD+TMD HEK293 cells ( ICD + TMD ) with MG-132 (5 μmol/liter) and immunoprecipitation with anti-FLAG antibody, non-reducing Western blotting showed dimerization of sortilin ICD+TMD using anti-sortilin antibody ( n = 3). Monomers, homodimers, and multimers are abbreviated as MO , D , and MU , respectively. IB , immunoblotting.

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Stable Transfection, Incubation, Concentration Assay, Immunoprecipitation

    Soluble sortilin forms homodimers. A and B , orientation of sortilin on the EV membrane was determined using EVs secreted from FLAG-sortilin HEK293 cells ( A ) and sortilin-3XFLAG HEK293 cells ( B ). EVs or their lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody, and FLAG-sortilin ( A ) or sortilin-3XFLAG ( B ) was detected by Western blotting with anti-FLAG antibody, showing that the extracellular domain of sortilin is located outside of EVs ( n = 3). C and D , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was detected in non-reducing ( C ) and reducing Western blotting ( D ), showing that they were homodimers and monomers, respectively ( n = 3). E , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was purified and detected in non-reducing Western blotting. IB , immunoblotting.
    Figure Legend Snippet: Soluble sortilin forms homodimers. A and B , orientation of sortilin on the EV membrane was determined using EVs secreted from FLAG-sortilin HEK293 cells ( A ) and sortilin-3XFLAG HEK293 cells ( B ). EVs or their lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody, and FLAG-sortilin ( A ) or sortilin-3XFLAG ( B ) was detected by Western blotting with anti-FLAG antibody, showing that the extracellular domain of sortilin is located outside of EVs ( n = 3). C and D , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was detected in non-reducing ( C ) and reducing Western blotting ( D ), showing that they were homodimers and monomers, respectively ( n = 3). E , soluble sortilin secreted by HEK293 cells overexpressing FLAG-sortilin Full and FLAG-sortilin ECD+TMD was purified and detected in non-reducing Western blotting. IB , immunoblotting.

    Techniques Used: Immunoprecipitation, Western Blot, Purification

    Sortilin S316E and sortilin wp increase dimerization in HEK293 cells, and the addition of propeptide decreases dimerization in the extracellular vesicles of FLAG-sortilin HEK293 cells. A , schematic of FLAG-sortilin WT, S316E, and wp. Serine 316 was replaced by glutamic acid in FLAG-sortilin S316E. Propeptide was removed in FLAG-sortilin wp. SP , signal peptide; PP , propeptide. B , S316E increased dimerization of sortilin in HEK293 cells ( n = 3). C , removal of propeptide increased dimerization of sortilin in HEK293 cells ( n = 3). D and E , addition of propeptide (100 nmol/liter) decreased dimerization of sortilin in the extracellular vesicles of FLAG-sortilin HEK293 cells ( E ), whereas a decrease in the cells was not observed ( D ) ( n = 2). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. Vec , vector; IB , immunoblotting.
    Figure Legend Snippet: Sortilin S316E and sortilin wp increase dimerization in HEK293 cells, and the addition of propeptide decreases dimerization in the extracellular vesicles of FLAG-sortilin HEK293 cells. A , schematic of FLAG-sortilin WT, S316E, and wp. Serine 316 was replaced by glutamic acid in FLAG-sortilin S316E. Propeptide was removed in FLAG-sortilin wp. SP , signal peptide; PP , propeptide. B , S316E increased dimerization of sortilin in HEK293 cells ( n = 3). C , removal of propeptide increased dimerization of sortilin in HEK293 cells ( n = 3). D and E , addition of propeptide (100 nmol/liter) decreased dimerization of sortilin in the extracellular vesicles of FLAG-sortilin HEK293 cells ( E ), whereas a decrease in the cells was not observed ( D ) ( n = 2). Monomers and homodimers of high and low molecular weight are abbreviated as MO , D(HMW) , and D(LMW) , respectively. Vec , vector; IB , immunoblotting.

    Techniques Used: Molecular Weight, Plasmid Preparation

    6) Product Images from "Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain *"

    Article Title: Antigen-specific Proteolysis by Hybrid Antibodies Containing Promiscuous Proteolytic Light Chains Paired with an Antigen-binding Heavy Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.011858

    Serine protease inhibitor effect and kinetics of Glu-Ala-Arg-AMC hydrolysis. A , phosphonate diester hapten inhibition of FLAG-E2 hydrolysis. Reducing SDS gels showing FLAG-E2 (1 n m ) incubated with diluent ( lane 1 ) or hybrid IgG HK14 (0.5 μ m ) in
    Figure Legend Snippet: Serine protease inhibitor effect and kinetics of Glu-Ala-Arg-AMC hydrolysis. A , phosphonate diester hapten inhibition of FLAG-E2 hydrolysis. Reducing SDS gels showing FLAG-E2 (1 n m ) incubated with diluent ( lane 1 ) or hybrid IgG HK14 (0.5 μ m ) in

    Techniques Used: Protease Inhibitor, Inhibition, Incubation

    Hydrolytic specificity of hybrid IgG: failure of the light chain alone to hydrolyze E2 proteins. Hydrolysis of FLAG-E2 ( left ) or GST-E2 ( right ) by hybrid IgG HK14 (1 μ m, stippled bars ) or light chain HK14 alone (2 μ m, solid bars ) was measured
    Figure Legend Snippet: Hydrolytic specificity of hybrid IgG: failure of the light chain alone to hydrolyze E2 proteins. Hydrolysis of FLAG-E2 ( left ) or GST-E2 ( right ) by hybrid IgG HK14 (1 μ m, stippled bars ) or light chain HK14 alone (2 μ m, solid bars ) was measured

    Techniques Used:

    Characteristics of E2 fusion protein hydrolysis by hybrid HK14. A , time dependence of FLAG-E2 hydrolysis. Hydrolysis of FLAG-E2 (1 n m ) by the hybrid IgG (0.5 μ m ) was measured as described in the legend to B . Mean ± S.D. of two reactions
    Figure Legend Snippet: Characteristics of E2 fusion protein hydrolysis by hybrid HK14. A , time dependence of FLAG-E2 hydrolysis. Hydrolysis of FLAG-E2 (1 n m ) by the hybrid IgG (0.5 μ m ) was measured as described in the legend to B . Mean ± S.D. of two reactions

    Techniques Used:

    E2 fusion protein cleavage regions. A , reducing SDS gel showing reaction mixtures of FLAG-E2 (1 n m ) incubated with wild type IgG CBH-7 or hybrid IgG HK14 (0.5 μ m ) for 48 h and stained with anti-E2 monoclonal Ab (respectively, lanes 1 and 2 ) or
    Figure Legend Snippet: E2 fusion protein cleavage regions. A , reducing SDS gel showing reaction mixtures of FLAG-E2 (1 n m ) incubated with wild type IgG CBH-7 or hybrid IgG HK14 (0.5 μ m ) for 48 h and stained with anti-E2 monoclonal Ab (respectively, lanes 1 and 2 ) or

    Techniques Used: SDS-Gel, Incubation, Staining

    Hydrolytic specificity of hybrid IgG: failure to hydrolyze irrelevant proteins and noncovalent E2 binding activity. A , substrate specificity. Reducing SDS gels showing reaction mixtures of hybrid IgG HK14 (0.5 μ m ) or diluent and FLAG-E2 (respectively,
    Figure Legend Snippet: Hydrolytic specificity of hybrid IgG: failure to hydrolyze irrelevant proteins and noncovalent E2 binding activity. A , substrate specificity. Reducing SDS gels showing reaction mixtures of hybrid IgG HK14 (0.5 μ m ) or diluent and FLAG-E2 (respectively,

    Techniques Used: Binding Assay, Activity Assay

    7) Product Images from "Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly"

    Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

    Journal: Brain

    doi: 10.1093/brain/awx203

    Analysis of AKT3 activity in vitro. ( A ) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. ( B ) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. ( C ) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed FLAG-tagged wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.
    Figure Legend Snippet: Analysis of AKT3 activity in vitro. ( A ) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. ( B ) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. ( C ) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD ( n = 4), P -values were determined using Student’s t -test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed FLAG-tagged wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.

    Techniques Used: Activity Assay, In Vitro, Binding Assay, Derivative Assay, Variant Assay, Mutagenesis, Ex Vivo, Kinase Assay, Western Blot, Quantitation Assay, Dot Blot, Incubation

    8) Product Images from "PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy"

    Article Title: PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy

    Journal: Cell Research

    doi: 10.1038/s41422-018-0056-0

    PTEN-L keeps Parkin in closed conformation by enhancing the interaction of Parkin UBL and RING1 domains in a protein phosphatase activity-dependent manner. a Construction of PTEN-L truncations. PTEN-L contains an ATR region, a phosphatase domain and a C-terminal region with a C2 domain and a C-Tail domain. PTEN-L-C297S is a dual lipid-protein phosphatase-defective mutant, while PTEN-L-G302R is a lipid phosphatase-defective mutant. b , c HEK293T cells transfected with GFP-Parkin and different constructs of Flag-tagged PTEN-L were treated without or with CCCP (5 µM) for 4 h. PTEN-L was immunoprecipitated with anti-Flag beads followed by immunoblotting for GFP and Flag. d Construction of Flag-tagged Parkin truncations, including Parkin-FL (full length) and truncated Parkin constructs: UBL-R0, Exon4, R1-IBR-R2, UBL-R1-IBR-R2 (deletion of R0 domain) and IBR-R2. e HEK293T cells transfected with GFP-PTEN-L and different constructs of Flag-tagged Parkin were treated without or with CCCP (5 µM) for 4 h. PTEN-L was then immunoprecipitated with anti-GFP beads followed by immunoblotting for Flag and GFP. f HEK293T cells were transfected with Flag-PTEN-L or the two Flag-PTEN-L mutants, together with Parkin truncation mutants GFP-UBL and GST-RING1 (R1) or GST empty vector (pEBG). Cells were then treated with or without CCCP (20 µM) for 4 h. RING1 was immunoprecipitated with anti-GST beads followed by immunoblotting for GFP, Flag and GST. g YFP-Parkin-HeLa cells transiently transfected with mCherry-PTEN-L or the two mCherry-PTEN-L mutants were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm
    Figure Legend Snippet: PTEN-L keeps Parkin in closed conformation by enhancing the interaction of Parkin UBL and RING1 domains in a protein phosphatase activity-dependent manner. a Construction of PTEN-L truncations. PTEN-L contains an ATR region, a phosphatase domain and a C-terminal region with a C2 domain and a C-Tail domain. PTEN-L-C297S is a dual lipid-protein phosphatase-defective mutant, while PTEN-L-G302R is a lipid phosphatase-defective mutant. b , c HEK293T cells transfected with GFP-Parkin and different constructs of Flag-tagged PTEN-L were treated without or with CCCP (5 µM) for 4 h. PTEN-L was immunoprecipitated with anti-Flag beads followed by immunoblotting for GFP and Flag. d Construction of Flag-tagged Parkin truncations, including Parkin-FL (full length) and truncated Parkin constructs: UBL-R0, Exon4, R1-IBR-R2, UBL-R1-IBR-R2 (deletion of R0 domain) and IBR-R2. e HEK293T cells transfected with GFP-PTEN-L and different constructs of Flag-tagged Parkin were treated without or with CCCP (5 µM) for 4 h. PTEN-L was then immunoprecipitated with anti-GFP beads followed by immunoblotting for Flag and GFP. f HEK293T cells were transfected with Flag-PTEN-L or the two Flag-PTEN-L mutants, together with Parkin truncation mutants GFP-UBL and GST-RING1 (R1) or GST empty vector (pEBG). Cells were then treated with or without CCCP (20 µM) for 4 h. RING1 was immunoprecipitated with anti-GST beads followed by immunoblotting for GFP, Flag and GST. g YFP-Parkin-HeLa cells transiently transfected with mCherry-PTEN-L or the two mCherry-PTEN-L mutants were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Construct, Immunoprecipitation, Plasmid Preparation

    PTEN-L dephosphorylates ubiquitin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Whole-cell lysates were analyzed by immunoblotting. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Immunofluorescence staining against pSer65-Ub was performed and observed by fluorescent microscopy. pSer65-Ub (red), YFP-Parkin (green), Nucleus (DAPI, blue). Scale bar, 10 µm. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. e In vitro dephosphorylation assay. Purified pSer65-Ub was incubated with purified Flag-PTEN-L, Flag-PTEN-L-C297S or Flag-PTEN-L-G302R in phosphatase reaction buffer and ubiquitin phosphorylation level was evaluated by immunoblotting
    Figure Legend Snippet: PTEN-L dephosphorylates ubiquitin. a YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Whole-cell lysates were analyzed by immunoblotting. b YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (5 µM) for 3 h. Immunofluorescence staining against pSer65-Ub was performed and observed by fluorescent microscopy. pSer65-Ub (red), YFP-Parkin (green), Nucleus (DAPI, blue). Scale bar, 10 µm. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. d Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with O/A (25 nM and 250 nM) for indicated hours and immunoblotting was performed. e In vitro dephosphorylation assay. Purified pSer65-Ub was incubated with purified Flag-PTEN-L, Flag-PTEN-L-C297S or Flag-PTEN-L-G302R in phosphatase reaction buffer and ubiquitin phosphorylation level was evaluated by immunoblotting

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence, Staining, Microscopy, In Vitro, De-Phosphorylation Assay, Purification, Incubation

    PTEN-L disrupts the feedforward mechanism in mitophagy by targeting the pSer65-Ub chains. a In vitro dephosphorylation assay using purified pSer65-tetra-Ub. Purified Flag-PTEN-L was incubated with pSer65-tetra-Ub in the phosphatase reaction buffer for 1 h at 30 °C. Calf intestinal phosphatase (CIP) was used as a positive control. b In vitro dephosphorylation assay using purified pSer65-poly-Ub chains, following the same procedure in a . λPP was used as a positive control. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down by GFP beads and subjected to immunoblotting. d The MS/MS spectra of the ubiquitin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h and YFP-Parkin was pulled down with GFP beads. e pSer65-Ub was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. *** P
    Figure Legend Snippet: PTEN-L disrupts the feedforward mechanism in mitophagy by targeting the pSer65-Ub chains. a In vitro dephosphorylation assay using purified pSer65-tetra-Ub. Purified Flag-PTEN-L was incubated with pSer65-tetra-Ub in the phosphatase reaction buffer for 1 h at 30 °C. Calf intestinal phosphatase (CIP) was used as a positive control. b In vitro dephosphorylation assay using purified pSer65-poly-Ub chains, following the same procedure in a . λPP was used as a positive control. c YFP-Parkin-HeLa cells with PTEN-L stable expression or control vector were treated with CCCP (10 µM) for 4 h. YFP-Parkin was pulled down by GFP beads and subjected to immunoblotting. d The MS/MS spectra of the ubiquitin peptide containing phospho-Ser65. YFP-Parkin-HeLa cells were treated with CCCP (10 µM) for 4 h and YFP-Parkin was pulled down with GFP beads. e pSer65-Ub was quantified using MS-based relative quantification analysis in YFP-Parkin-HeLa cells with or without PTEN-L stable expression after CCCP (10 µM) and O/A (25 nM and 250 nM) treatment. Data are presented as mean ± SD from 3 independent experiments. *** P

    Techniques Used: In Vitro, De-Phosphorylation Assay, Purification, Incubation, Positive Control, Expressing, Plasmid Preparation, Mass Spectrometry

    PTEN-L resides at the outer mitochondrial membrane. a Domain structure of PTEN-L protein. b Cell fractionation was performed to isolate mitochondria in HeLa cells. Tim23 and GAPDH were used as mitochondrial and cytosolic markers, respectively. C cytosol, M mitochondria. c Relative ratios of PTEN-L and PTEN in cytosol and mitochondria were calculated with normalization of volume. d Topology assay showing PTEN-L localization at the OMM. Mitochondria were isolated from YFP-Parkin-HeLa cells stably expressing Flag-PTEN-L and treated with different doses of proteinase K and digitonin. e Immunogold EM. YFP-Parkin-HeLa cells stably expressing Flag-PTEN-L were treated without ( A , B ) or with CCCP (5 µM) ( C , D ) for 4 h. Cells were subjected to immunoelectron microscopy with anti-Flag antibody. Panels ( B ) and ( D ) are the magnified images of boxes in ( A ) and ( C ), respectively. Arrows indicate ER structure
    Figure Legend Snippet: PTEN-L resides at the outer mitochondrial membrane. a Domain structure of PTEN-L protein. b Cell fractionation was performed to isolate mitochondria in HeLa cells. Tim23 and GAPDH were used as mitochondrial and cytosolic markers, respectively. C cytosol, M mitochondria. c Relative ratios of PTEN-L and PTEN in cytosol and mitochondria were calculated with normalization of volume. d Topology assay showing PTEN-L localization at the OMM. Mitochondria were isolated from YFP-Parkin-HeLa cells stably expressing Flag-PTEN-L and treated with different doses of proteinase K and digitonin. e Immunogold EM. YFP-Parkin-HeLa cells stably expressing Flag-PTEN-L were treated without ( A , B ) or with CCCP (5 µM) ( C , D ) for 4 h. Cells were subjected to immunoelectron microscopy with anti-Flag antibody. Panels ( B ) and ( D ) are the magnified images of boxes in ( A ) and ( C ), respectively. Arrows indicate ER structure

    Techniques Used: Cell Fractionation, Isolation, Stable Transfection, Expressing, Immuno-Electron Microscopy

    PTEN-L prevents Parkin mitochondrial translocation. a YFP-Parkin-HeLa cells transiently transfected with Flag-PTEN-L or control vector were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), Tom20 (red), Flag-PTEN-L (cyan). Scale bar, 10 µm. b Percentage of cells with Parkin mitochondrial translocation was quantified by counting at least 300 cells. c Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (4 µM) for 30 and 90 min. YFP-Parkin (green). Scale bar, 10 µm. d Percentage of cells with Parkin mitochondrial translocation from c (CCCP 90 min) was quantified by counting at least 300 cells. e PTEN-L KO YFP-Parkin-HeLa cells were transiently transfected with mCherry-PTEN-L or control vector and treated with CCCP (4 µM) for 90 min. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm. Data in b , d are presented as mean ± SD from three independent experiments. ** P
    Figure Legend Snippet: PTEN-L prevents Parkin mitochondrial translocation. a YFP-Parkin-HeLa cells transiently transfected with Flag-PTEN-L or control vector were treated with CCCP (5 µM) for 2 h. YFP-Parkin (green), Tom20 (red), Flag-PTEN-L (cyan). Scale bar, 10 µm. b Percentage of cells with Parkin mitochondrial translocation was quantified by counting at least 300 cells. c Wild-type (WT) and PTEN-L KO YFP-Parkin-HeLa cells were treated with CCCP (4 µM) for 30 and 90 min. YFP-Parkin (green). Scale bar, 10 µm. d Percentage of cells with Parkin mitochondrial translocation from c (CCCP 90 min) was quantified by counting at least 300 cells. e PTEN-L KO YFP-Parkin-HeLa cells were transiently transfected with mCherry-PTEN-L or control vector and treated with CCCP (4 µM) for 90 min. YFP-Parkin (green), mCherry (red). Scale bar, 10 µm. Data in b , d are presented as mean ± SD from three independent experiments. ** P

    Techniques Used: Translocation Assay, Transfection, Plasmid Preparation

    9) Product Images from "Aryl hydrocarbon (Ah) receptor levels are selectively modulated by hsp90-associated immunophilin homolog XAP2"

    Article Title: Aryl hydrocarbon (Ah) receptor levels are selectively modulated by hsp90-associated immunophilin homolog XAP2

    Journal: Cell Stress & Chaperones

    doi:

    EYFP-XAP2 binds to hsp90 and to AhR/hsp90 complexes in COS-1 cells. Upper panels: pEYFP-XAP2-FLAG or pEYFP alone was transiently transfected in COS-1 cells, cytosol was isolated, and 150 μg of protein were resolved by SDS-PAGE and transferred to PVDF, followed by immunoblot analysis with XAP2 polyclonal antibodies or anti-GFP monoclonal antibodies. Antibodies were visualized with DAR-P and GAM-P, respectively. Lower panels: pEYFP-XAP2-FLAG or pEYFP were transiently transfected in COS-1 cells (left panel); pEYFP-XAP2-FLAG or pEYFP were transiently cotransfected with pcDNA3/βmAhR in COS-1 cells (right panel). In both experiments, cytosol was isolated and immunoabsorbed with the M2 affinity resin, and complexes were eluted with FLAG peptide and resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. pEYFP-XAP2-FLAG was visualized with the M2 antibody, AhR with the RPT1 antibody, and hsp90 with rabbit polyclonal antibodies against hsp84 and hsp86. M2 antibody was visualized with GAM-P, hsp90 with DAR-P, and AhR with GAM-P by ECL
    Figure Legend Snippet: EYFP-XAP2 binds to hsp90 and to AhR/hsp90 complexes in COS-1 cells. Upper panels: pEYFP-XAP2-FLAG or pEYFP alone was transiently transfected in COS-1 cells, cytosol was isolated, and 150 μg of protein were resolved by SDS-PAGE and transferred to PVDF, followed by immunoblot analysis with XAP2 polyclonal antibodies or anti-GFP monoclonal antibodies. Antibodies were visualized with DAR-P and GAM-P, respectively. Lower panels: pEYFP-XAP2-FLAG or pEYFP were transiently transfected in COS-1 cells (left panel); pEYFP-XAP2-FLAG or pEYFP were transiently cotransfected with pcDNA3/βmAhR in COS-1 cells (right panel). In both experiments, cytosol was isolated and immunoabsorbed with the M2 affinity resin, and complexes were eluted with FLAG peptide and resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. pEYFP-XAP2-FLAG was visualized with the M2 antibody, AhR with the RPT1 antibody, and hsp90 with rabbit polyclonal antibodies against hsp84 and hsp86. M2 antibody was visualized with GAM-P, hsp90 with DAR-P, and AhR with GAM-P by ECL

    Techniques Used: Transfection, Isolation, SDS Page

    ). (B) COS-1 cells were transiently transfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control), and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. Hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [ 125 I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [ 125 I]-SAM
    Figure Legend Snippet: ). (B) COS-1 cells were transiently transfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control), and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. Hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [ 125 I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [ 125 I]-SAM

    Techniques Used: Transfection, Isolation, SDS Page

    XAP2 specifically enhances the level of AhR in COS-1 cells when compared to other hsp90-binding TPR-containing proteins. COS-1 cells were transfected in 6 well dishes with 1 μg of pcDNA3/βmAhR and 0, 0.2, 0.4, 0.6, 0.8, or 1.0 μg of pCI/XAP2, pCI/FKBP52, pCMV6/PP5-FLAG, or pCMV6/PP5-TPR-FLAG (shaded triangle) and brought to a total of 2 μg vector/dish with pCI vector. (A) Total cell lysates were isolated, and 75 μg of each lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. XAP2 and FKBP52 were detected with XAP2 polyclonal and FKBP52 monoclonal antibodies. PP5-TPR and PP5-TPR-FLAG were detected with anti-FLAG M2 antibody. AhR was detected with the anti-AhR monoclonal antibody RPT1. XAP2 was visualized with [ 125 I]-DAR, and the AhR, FKBP52, PP5-FLAG, and PP5-TPR-FLAG were visualized with [125I]-SAM. (B) The graph depicts the fold change in AhR levels obtained in the presence of TPR-containing proteins after phosphorimaging of the blots. This experiment has been repeated 3 times with essentially the same results
    Figure Legend Snippet: XAP2 specifically enhances the level of AhR in COS-1 cells when compared to other hsp90-binding TPR-containing proteins. COS-1 cells were transfected in 6 well dishes with 1 μg of pcDNA3/βmAhR and 0, 0.2, 0.4, 0.6, 0.8, or 1.0 μg of pCI/XAP2, pCI/FKBP52, pCMV6/PP5-FLAG, or pCMV6/PP5-TPR-FLAG (shaded triangle) and brought to a total of 2 μg vector/dish with pCI vector. (A) Total cell lysates were isolated, and 75 μg of each lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. XAP2 and FKBP52 were detected with XAP2 polyclonal and FKBP52 monoclonal antibodies. PP5-TPR and PP5-TPR-FLAG were detected with anti-FLAG M2 antibody. AhR was detected with the anti-AhR monoclonal antibody RPT1. XAP2 was visualized with [ 125 I]-DAR, and the AhR, FKBP52, PP5-FLAG, and PP5-TPR-FLAG were visualized with [125I]-SAM. (B) The graph depicts the fold change in AhR levels obtained in the presence of TPR-containing proteins after phosphorimaging of the blots. This experiment has been repeated 3 times with essentially the same results

    Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Isolation, SDS Page

    YFP-XAP2/Flag and YFP-XAP2/Flag G272D are localized in both the cytoplasmic and nuclear compartments. NIH 3T3 cells were transiently transfected with pEYFP (top panels), pEYFP-XAP2/Flag (middle panels), or pEYFP-XAP2 G272D (bottom panels). After 24 h, the transfected cells were visualized by either epifluorecence (left panels) or scanning confocal microscopy (right panels). The confocal images represent a horizontal midsectional view of the cell. Each image is representative of the population of transfected cells
    Figure Legend Snippet: YFP-XAP2/Flag and YFP-XAP2/Flag G272D are localized in both the cytoplasmic and nuclear compartments. NIH 3T3 cells were transiently transfected with pEYFP (top panels), pEYFP-XAP2/Flag (middle panels), or pEYFP-XAP2 G272D (bottom panels). After 24 h, the transfected cells were visualized by either epifluorecence (left panels) or scanning confocal microscopy (right panels). The confocal images represent a horizontal midsectional view of the cell. Each image is representative of the population of transfected cells

    Techniques Used: Transfection, Confocal Microscopy

    Mutants G272D, G272E, and F288A are unable to interact with the AhR in the absence of hsp90 in a cell-free system, and a conserved glycine in the XAP2 TPR complex is required for assembly of XAP2 in AhR-hsp90 complexes in COS-1 cells. (A) XAP2-FLAG, XAP2-FLAG-TPR mutants, or control (RL) were labeled with [ 35 S] methionine in RL independently, immunoabsorbed with M2 resin, and eluted with FLAG peptide. mAhR was generated in RL (unlabeled) and immunoabsorbed with RPT9/protein G sepharose or with murine IgG/protein G sepharose (control, last 2 lanes). The AhR immunoabsorption was washed in PBS to remove hsp90. RPT9-sepharose-mAhR was mixed with eluted XAP2-FLAG or XAP2-FLAG TPR mutants, and complexes washed, resolved by SDS-PAGE, and transferred to PVDF membrane. Top panel: mAhR visualized with RPT1 and [ 125 I]-SAM by autoradiography. Middle panel: XAP2-FLAG visualized by autoradiography. (B) COS-1 cells were transiently cotransfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control) and pcDNA3/_mAhR, and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. AhR was visualized with RPT1 and [ 125 I]-SAM, hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [ 125 I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [ 125 I]-SAM. >
    Figure Legend Snippet: Mutants G272D, G272E, and F288A are unable to interact with the AhR in the absence of hsp90 in a cell-free system, and a conserved glycine in the XAP2 TPR complex is required for assembly of XAP2 in AhR-hsp90 complexes in COS-1 cells. (A) XAP2-FLAG, XAP2-FLAG-TPR mutants, or control (RL) were labeled with [ 35 S] methionine in RL independently, immunoabsorbed with M2 resin, and eluted with FLAG peptide. mAhR was generated in RL (unlabeled) and immunoabsorbed with RPT9/protein G sepharose or with murine IgG/protein G sepharose (control, last 2 lanes). The AhR immunoabsorption was washed in PBS to remove hsp90. RPT9-sepharose-mAhR was mixed with eluted XAP2-FLAG or XAP2-FLAG TPR mutants, and complexes washed, resolved by SDS-PAGE, and transferred to PVDF membrane. Top panel: mAhR visualized with RPT1 and [ 125 I]-SAM by autoradiography. Middle panel: XAP2-FLAG visualized by autoradiography. (B) COS-1 cells were transiently cotransfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control) and pcDNA3/_mAhR, and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. AhR was visualized with RPT1 and [ 125 I]-SAM, hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [ 125 I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [ 125 I]-SAM. >

    Techniques Used: Labeling, Generated, SDS Page, Autoradiography, Isolation

    10) Product Images from "Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability"

    Article Title: Genetic and pharmacological inhibition of TTK impairs pancreatic cancer cell line growth by inducing lethal chromosomal instability

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174863

    Usp16 is a TTK phosphorylation substrate. (A) In vitro kinase assay measuring TTK dependent phosphorylation by 32 P incorporation measured by liquid scintillation counts. Representative of 2 independent experiments. (B) Exogenously expressed FLAG-Usp16 was immunoprecipitated from DMSO and AZ3146 treated mitotic 293FT cells, digested with trypsin and enriched for phosphopeptides. Phosphorylated residues of Usp16 were identified by mass spectrometry. Spectral counts of representative individual experiments are shown. (C) Immunoblot analysis of 293FT cells transiently transfected with control GFP, GFP-Usp16, GFP-Usp16 3xA (phosphodeficient mutant) or GFP-Usp16 3xE (phosphomimetic mutant) and treated with control DMSO or MG-132. (D) Densitometry of (C). (E) RT-PCR of Usp16 using 2 independent Taqman probes from cells used in (C), normalized to β-actin and represented as percent of WT-Usp16.
    Figure Legend Snippet: Usp16 is a TTK phosphorylation substrate. (A) In vitro kinase assay measuring TTK dependent phosphorylation by 32 P incorporation measured by liquid scintillation counts. Representative of 2 independent experiments. (B) Exogenously expressed FLAG-Usp16 was immunoprecipitated from DMSO and AZ3146 treated mitotic 293FT cells, digested with trypsin and enriched for phosphopeptides. Phosphorylated residues of Usp16 were identified by mass spectrometry. Spectral counts of representative individual experiments are shown. (C) Immunoblot analysis of 293FT cells transiently transfected with control GFP, GFP-Usp16, GFP-Usp16 3xA (phosphodeficient mutant) or GFP-Usp16 3xE (phosphomimetic mutant) and treated with control DMSO or MG-132. (D) Densitometry of (C). (E) RT-PCR of Usp16 using 2 independent Taqman probes from cells used in (C), normalized to β-actin and represented as percent of WT-Usp16.

    Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Mass Spectrometry, Transfection, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    11) Product Images from "IL-17 receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling"

    Article Title: IL-17 receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling

    Journal: Nature immunology

    doi: 10.1038/s41590-018-0071-9

    IL-17A induces distinct Act1-RNPs in the nucleus and cytoplasmic granules. a . Confocal imaging of Act1-GFP in HeLa Tet-On stable cell line induced by doxycycline for 24 hours followed by IL-17A stimulation. Nuclei stained with DAPI in blue. Bar graph shows the mean and s.d. of percentages (n=3 independent plates of cells) of cells that display Act1 in the cytoplasmic granules or nuclear localization. 50 cells per plate were analyzed for quantification. b . Confocal imaging of HeLa cells co-transfected with GFP- or RPF-tagged expression constructs as indicated. Nuclei stained with DAPI in blue. Bar graph shows the mean and s.d. of percentages (n=3 independent plates of cells) of cells with co-localization of the two expressing proteins [Act1/Dcp1; Act1/TIA1 and Dcp1/IRAK1(IL-1R associated kinase 1)]. 50 cells per plate were analyzed for quantification. c. Lysates from primary MEFs (isolated from LSL–HA–Act1 knock-in mice) stimulated with IL-17A (50 ng/ml), either left untreated or treated with RNase A, were immunoprecipitated (IP) using anti-HA (for endogenous HA-Act1) followed by Western blot analysis with the indicated antibodies. The data are representative of three independent experiments. d. Bar graph shows the mean and s.d. (n=3 independent plates of cells) of percentages of cells with co-localization of Dcp1 with full-length Act1 or Act1 deletion mutants ( Supplemental. Fig. 1g ). 50 expressing cells per plate were analyzed for quantification. e . Act1 −/− MEFs reconstituted by retroviral infection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant were treated with IL-17A (50 ng/ml). The cell lysates were immunoprecipitated (IP) with anti-FLAG followed by Western blot analysis using antibodies as indicated. All data are representative of two independent experiments. The exact P value for each comparison is provided on the graph or as p value
    Figure Legend Snippet: IL-17A induces distinct Act1-RNPs in the nucleus and cytoplasmic granules. a . Confocal imaging of Act1-GFP in HeLa Tet-On stable cell line induced by doxycycline for 24 hours followed by IL-17A stimulation. Nuclei stained with DAPI in blue. Bar graph shows the mean and s.d. of percentages (n=3 independent plates of cells) of cells that display Act1 in the cytoplasmic granules or nuclear localization. 50 cells per plate were analyzed for quantification. b . Confocal imaging of HeLa cells co-transfected with GFP- or RPF-tagged expression constructs as indicated. Nuclei stained with DAPI in blue. Bar graph shows the mean and s.d. of percentages (n=3 independent plates of cells) of cells with co-localization of the two expressing proteins [Act1/Dcp1; Act1/TIA1 and Dcp1/IRAK1(IL-1R associated kinase 1)]. 50 cells per plate were analyzed for quantification. c. Lysates from primary MEFs (isolated from LSL–HA–Act1 knock-in mice) stimulated with IL-17A (50 ng/ml), either left untreated or treated with RNase A, were immunoprecipitated (IP) using anti-HA (for endogenous HA-Act1) followed by Western blot analysis with the indicated antibodies. The data are representative of three independent experiments. d. Bar graph shows the mean and s.d. (n=3 independent plates of cells) of percentages of cells with co-localization of Dcp1 with full-length Act1 or Act1 deletion mutants ( Supplemental. Fig. 1g ). 50 expressing cells per plate were analyzed for quantification. e . Act1 −/− MEFs reconstituted by retroviral infection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant were treated with IL-17A (50 ng/ml). The cell lysates were immunoprecipitated (IP) with anti-FLAG followed by Western blot analysis using antibodies as indicated. All data are representative of two independent experiments. The exact P value for each comparison is provided on the graph or as p value

    Techniques Used: Imaging, Stable Transfection, Staining, Transfection, Expressing, Construct, Isolation, Knock-In, Mouse Assay, Immunoprecipitation, Western Blot, Infection, Mutagenesis

    Act1-RNA binding to 3’UTR inhibits decapping through TBK1-mediated phosphorylation of Dcp1. a. Cap (m7GDP) labeled reporter RNAs [ Cxcl 220 and mutant without stem-loop C ( Cxcl 220-stem-loop C mutant) ] were subjected to decapping assay using purified Dcp1/Dcp2 with increasing amounts of purified Act1 WT or Act1 ΔSEFIR in the presence or absence of TBK1 inhibitor (MRT67307). EDTA and boiling were included as negative controls to inactivate Dcp1/Dcp2 enzymatic activity. Radiograms were quantified by densitometry. b. Graph indicates the decapping activity calculated by quantifying the released Cap as a percentage of the amount of Cap catalyzed by purified Dcp1/2. c. Cell lysates from Act1 −/− MEFs with and without reconstitution of wild-type Act1 (Act1-WT and Act1-KO) with or without the presence of TBK1 inhibitor (MRT67307) were immunoprecipitated (IP) with anti-TBK1 followed by Western blot analysis using antibodies as indicated. d. Cell lysates from untreated and IL-17A-treated HeLa cells were immunoprecipitated (IP) with anti-Act1 followed by Western blot analysis using antibodies as indicated. e. In vitro kinase assay of Dcp1 by recombinant TBK1 using Dcp1-immunoprecipitates from HeLa cells transfected with FLAG-tagged Dcp1. f. Wild-type (WT), IKKi-deficient (IKKi KO) and TBK1-deficient (TBK1 KO) MEFs pretreated with TNF for 1 hour, were untreated or stimulated with IL-17A. The mRNA and protein levels were then analyzed by RT-PCR (Top) and Elisa (Bottom), respectively (n=3 independent plates of cells). Bar graph shows mean and s.d. of independent plates of cells (f). The exact P value for each comparison is provided in the graph or as p value
    Figure Legend Snippet: Act1-RNA binding to 3’UTR inhibits decapping through TBK1-mediated phosphorylation of Dcp1. a. Cap (m7GDP) labeled reporter RNAs [ Cxcl 220 and mutant without stem-loop C ( Cxcl 220-stem-loop C mutant) ] were subjected to decapping assay using purified Dcp1/Dcp2 with increasing amounts of purified Act1 WT or Act1 ΔSEFIR in the presence or absence of TBK1 inhibitor (MRT67307). EDTA and boiling were included as negative controls to inactivate Dcp1/Dcp2 enzymatic activity. Radiograms were quantified by densitometry. b. Graph indicates the decapping activity calculated by quantifying the released Cap as a percentage of the amount of Cap catalyzed by purified Dcp1/2. c. Cell lysates from Act1 −/− MEFs with and without reconstitution of wild-type Act1 (Act1-WT and Act1-KO) with or without the presence of TBK1 inhibitor (MRT67307) were immunoprecipitated (IP) with anti-TBK1 followed by Western blot analysis using antibodies as indicated. d. Cell lysates from untreated and IL-17A-treated HeLa cells were immunoprecipitated (IP) with anti-Act1 followed by Western blot analysis using antibodies as indicated. e. In vitro kinase assay of Dcp1 by recombinant TBK1 using Dcp1-immunoprecipitates from HeLa cells transfected with FLAG-tagged Dcp1. f. Wild-type (WT), IKKi-deficient (IKKi KO) and TBK1-deficient (TBK1 KO) MEFs pretreated with TNF for 1 hour, were untreated or stimulated with IL-17A. The mRNA and protein levels were then analyzed by RT-PCR (Top) and Elisa (Bottom), respectively (n=3 independent plates of cells). Bar graph shows mean and s.d. of independent plates of cells (f). The exact P value for each comparison is provided in the graph or as p value

    Techniques Used: RNA Binding Assay, Labeling, Mutagenesis, Purification, Activity Assay, Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Recombinant, Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Act1 forms distinct RNPs with Dcp1, Dcp2, SF2 and HuR. a. Act1 −/− MEFs reconstituted by retroviral infection with either wild-type Act1 (WT) or ΔSEFIR1 were pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 60 min followed by RNA immunoprecipitation with anti-SF2 or anti-HuR, and RT-PCR analyses (n=3 independent plates of cells) of the indicated mRNAs. The presented are the relative values normalized against IgG control. b. Schematic representation of the mouse CXCL1 3’UTR. HuR, Act1 and SF2 binding regions are indicated. c . Act1-SEFIR and SF2 RNA binding competition was performed using probe Cxcl1 220 II (as indicated in b) . Graph indicates the dissociation of SF2- Cxcl1 220 II upon incubation with increasing amounts of Act1-SEFIR, which was calculated by quantifying the remaining SF2- Cxcl1 220 II complex in the presence of indicated amounts of Act1-SEFIR. d. In vitro kinase assay of purified recombinant IKKi using purified recombinant SF2 as a substrate. e. The simultaneous binding of purified recombinant HuR and Act1-SEFIR to Cxcl1 3’UTR was examined by REMSA using Cxcl1 220 and Cxcl1 220 II as probes. The co-binding Act1-HuR was observed with probe Cxcl1 -220, but not Cxcl1 -220 II. f. UV-absorbance profile of RNP and polysome complexes separated on a sucrose density gradient into different fractions as indicated. g. Cytoplasmic extracts of Act1 −/− MEFs reconstituted by retroviral infection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant, pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 90 min, were fractionated through a 10–50% sucrose gradient and analyzed by Western blot analyses with the indicated antibodies. h. Cxcl1 mRNAs from translation-active pools and translation-inactive pools from f were analyzed by RT-PCR and normalized to β-actin. Graph shows the ratios of mRNAs from translation-active/inactive pools (n=3 independent plates of cells). All data are representative of three independent experiments. Bar graph shows mean and s.d. of independent plates of cells ( a, h ). The exact P value for each comparison is provided in the graph or as p value
    Figure Legend Snippet: Act1 forms distinct RNPs with Dcp1, Dcp2, SF2 and HuR. a. Act1 −/− MEFs reconstituted by retroviral infection with either wild-type Act1 (WT) or ΔSEFIR1 were pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 60 min followed by RNA immunoprecipitation with anti-SF2 or anti-HuR, and RT-PCR analyses (n=3 independent plates of cells) of the indicated mRNAs. The presented are the relative values normalized against IgG control. b. Schematic representation of the mouse CXCL1 3’UTR. HuR, Act1 and SF2 binding regions are indicated. c . Act1-SEFIR and SF2 RNA binding competition was performed using probe Cxcl1 220 II (as indicated in b) . Graph indicates the dissociation of SF2- Cxcl1 220 II upon incubation with increasing amounts of Act1-SEFIR, which was calculated by quantifying the remaining SF2- Cxcl1 220 II complex in the presence of indicated amounts of Act1-SEFIR. d. In vitro kinase assay of purified recombinant IKKi using purified recombinant SF2 as a substrate. e. The simultaneous binding of purified recombinant HuR and Act1-SEFIR to Cxcl1 3’UTR was examined by REMSA using Cxcl1 220 and Cxcl1 220 II as probes. The co-binding Act1-HuR was observed with probe Cxcl1 -220, but not Cxcl1 -220 II. f. UV-absorbance profile of RNP and polysome complexes separated on a sucrose density gradient into different fractions as indicated. g. Cytoplasmic extracts of Act1 −/− MEFs reconstituted by retroviral infection with either FLAG-tagged mouse wild-type Act1 (WT) or SEFIR1 deletion mutant, pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 90 min, were fractionated through a 10–50% sucrose gradient and analyzed by Western blot analyses with the indicated antibodies. h. Cxcl1 mRNAs from translation-active pools and translation-inactive pools from f were analyzed by RT-PCR and normalized to β-actin. Graph shows the ratios of mRNAs from translation-active/inactive pools (n=3 independent plates of cells). All data are representative of three independent experiments. Bar graph shows mean and s.d. of independent plates of cells ( a, h ). The exact P value for each comparison is provided in the graph or as p value

    Techniques Used: Infection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, RNA Binding Assay, Incubation, In Vitro, Kinase Assay, Purification, Recombinant, Mutagenesis, Western Blot

    Act1 directly binds to the CXCL1 3’UTR through the SEFIR domain a. The structure model of Act1-SEFIR. Blue and Red: positive and negative electrostatic potential. b. Binding of purified recombinant Act1 SEFIR, IL-17RA SEFIR and His-MBP to the Cxcl1 3’UTR and Gpx4 3’UTR by REMSA. Graph indicates the apparent Kd of Cxcl1 3’UTR ( Cxcl1 220 as shown in Fig. 3a ). Graph shows mean and s.d. c . REMSA of purified recombinant Act1 SEFIR and SEFIR point mutants to the Cxcl1 3’UTR ( Cxcl1 220). d. FLAG-tagged Act1 WT and Act1 mutants were transfected into MEFs with V5-tagged IL-17RA. The cell lysates were immunoprecipated (IP) using anti-V5 followed by Western blot analyses using the indicated antibodies. e . Act1 −/− MEFs transfected with Act1 wild-type (Act1-WT) and Act1 mutants were treated with IL-17A for the indicated times. The cell lysates were analyzed by Western blotting with the indicated antibodies. f. Act1 −/− MEFs transfected with vector (Act1-KO), Act1 wild-type (Act1-WT) and Act1 mutants pretreated with TNF for 1 hour, either left untreated or stimulated with IL-17A. The mRNA and protein levels were analyzed by RT-PCR (Top) and Elisa (Bottom), respectively (n=3 independent plates of cells). g. Act1 −/− MEFs transfected with vector (Act1-KO), Act1 wild-type (Act1-WT) and Act1 mutants were pre-treated with TNF for 1 hour and then treated with Actinomycin D alone (NT) or in the presence of IL-17A for 25, 50, 70, and 100min, followed by RT-PCR analysis (n=3 independent plates of cells). The indicated mRNA levels were normalized to GAPDH and presented as half-life. h . Act1 −/− MEFs transfected with vector (Act1-KO), flag-tagged Act1 wild-type (Act1-WT) and Act1 mutants were pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 60 min followed by RNA immunoprecipitation with anti-FLAG and RT-PCR analyses of the indicated mRNAs (n=3 independent plates of cells). The presented are the relative values to levels from IgG immunoprecipitation. Bar graph shows mean and s.d. of independent plates of cells ( f-h ). The exact P value for each comparison (Act1-WT versus Act1 mutants) is provided in the graph or as p value
    Figure Legend Snippet: Act1 directly binds to the CXCL1 3’UTR through the SEFIR domain a. The structure model of Act1-SEFIR. Blue and Red: positive and negative electrostatic potential. b. Binding of purified recombinant Act1 SEFIR, IL-17RA SEFIR and His-MBP to the Cxcl1 3’UTR and Gpx4 3’UTR by REMSA. Graph indicates the apparent Kd of Cxcl1 3’UTR ( Cxcl1 220 as shown in Fig. 3a ). Graph shows mean and s.d. c . REMSA of purified recombinant Act1 SEFIR and SEFIR point mutants to the Cxcl1 3’UTR ( Cxcl1 220). d. FLAG-tagged Act1 WT and Act1 mutants were transfected into MEFs with V5-tagged IL-17RA. The cell lysates were immunoprecipated (IP) using anti-V5 followed by Western blot analyses using the indicated antibodies. e . Act1 −/− MEFs transfected with Act1 wild-type (Act1-WT) and Act1 mutants were treated with IL-17A for the indicated times. The cell lysates were analyzed by Western blotting with the indicated antibodies. f. Act1 −/− MEFs transfected with vector (Act1-KO), Act1 wild-type (Act1-WT) and Act1 mutants pretreated with TNF for 1 hour, either left untreated or stimulated with IL-17A. The mRNA and protein levels were analyzed by RT-PCR (Top) and Elisa (Bottom), respectively (n=3 independent plates of cells). g. Act1 −/− MEFs transfected with vector (Act1-KO), Act1 wild-type (Act1-WT) and Act1 mutants were pre-treated with TNF for 1 hour and then treated with Actinomycin D alone (NT) or in the presence of IL-17A for 25, 50, 70, and 100min, followed by RT-PCR analysis (n=3 independent plates of cells). The indicated mRNA levels were normalized to GAPDH and presented as half-life. h . Act1 −/− MEFs transfected with vector (Act1-KO), flag-tagged Act1 wild-type (Act1-WT) and Act1 mutants were pre-treated with TNF for 1 hour and then treated with IL-17A for 0 and 60 min followed by RNA immunoprecipitation with anti-FLAG and RT-PCR analyses of the indicated mRNAs (n=3 independent plates of cells). The presented are the relative values to levels from IgG immunoprecipitation. Bar graph shows mean and s.d. of independent plates of cells ( f-h ). The exact P value for each comparison (Act1-WT versus Act1 mutants) is provided in the graph or as p value

    Techniques Used: Binding Assay, Purification, Recombinant, Transfection, Western Blot, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

    12) Product Images from "Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex *"

    Article Title: Claudin-2 Forms Homodimers and Is a Component of a High Molecular Weight Protein Complex *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.195578

    Claudins 1, 2, and 7 form discrete oligomers in MDCK cells. A , sucrose gradient-enriched plasma membrane fractions from MDCK II cells were extracted with 1% DDM, electrophoresed in BN-PAGE, transferred to PVDF membrane, and immunoblotted for cldn1, -2, and -7 and occludin ( ocln ); molecular weights determined by comparison with native mark molecular weight markers (Invitrogen) reveal that claudins but not occludin migrate consistent with being either dimers or trimers. B , stably transfected MDCK II cells were induced to express cldn2 or cldn4, extracted with 1% DDM without (−) or with (+) the addition of 1% SDS, and electrophoresed in BN-PAGE. SDS-treated samples reveal position of monomer; DDM-extracted cldn2 but not cldn4 migrates as a dimer. C , HEK cells were transfected with FLAG-cldn 2 ( lane 1 ), His-cldn2 ( lane 2 ), co-transfected with both His- and FLAG-cldn2 ( lane 3 ), separately transfected with His- and FLAG-cldn2, and then co-plated ( lane 4 ); all cells were extracted with 1% DDM, and supernatants from separately transfected cells were mixed after extraction ( lane 5 ). Aliquots of DDM extracts were electrophoresed in SDS-PAGE and immunoblotted ( IB ) for cldn2 ( upper left ) and FLAG epitope ( lower left ). Remaining lysates were applied to Talon resin, washed, and eluted with imidazole; eluates were subjected to SDS-PAGE and immunoblotted with cldn2 ( upper right ) and FLAG antibodies ( lower right ). FLAG-cldn2 is retained on Talon resin when co-transfected with His-cldn2, consistent with homodimer formation during biosynthesis or trafficking but not via intercellular interactions.
    Figure Legend Snippet: Claudins 1, 2, and 7 form discrete oligomers in MDCK cells. A , sucrose gradient-enriched plasma membrane fractions from MDCK II cells were extracted with 1% DDM, electrophoresed in BN-PAGE, transferred to PVDF membrane, and immunoblotted for cldn1, -2, and -7 and occludin ( ocln ); molecular weights determined by comparison with native mark molecular weight markers (Invitrogen) reveal that claudins but not occludin migrate consistent with being either dimers or trimers. B , stably transfected MDCK II cells were induced to express cldn2 or cldn4, extracted with 1% DDM without (−) or with (+) the addition of 1% SDS, and electrophoresed in BN-PAGE. SDS-treated samples reveal position of monomer; DDM-extracted cldn2 but not cldn4 migrates as a dimer. C , HEK cells were transfected with FLAG-cldn 2 ( lane 1 ), His-cldn2 ( lane 2 ), co-transfected with both His- and FLAG-cldn2 ( lane 3 ), separately transfected with His- and FLAG-cldn2, and then co-plated ( lane 4 ); all cells were extracted with 1% DDM, and supernatants from separately transfected cells were mixed after extraction ( lane 5 ). Aliquots of DDM extracts were electrophoresed in SDS-PAGE and immunoblotted ( IB ) for cldn2 ( upper left ) and FLAG epitope ( lower left ). Remaining lysates were applied to Talon resin, washed, and eluted with imidazole; eluates were subjected to SDS-PAGE and immunoblotted with cldn2 ( upper right ) and FLAG antibodies ( lower right ). FLAG-cldn2 is retained on Talon resin when co-transfected with His-cldn2, consistent with homodimer formation during biosynthesis or trafficking but not via intercellular interactions.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Molecular Weight, Stable Transfection, Transfection, SDS Page, FLAG-tag

    13) Product Images from "The role of Cas8 in type I CRISPR interference"

    Article Title: The role of Cas8 in type I CRISPR interference

    Journal: Bioscience Reports

    doi: 10.1042/BSR20150043

    Interaction of Cas8 and Cas8′ with Cas5–Cas7 ( A ) Coomassie stained SDS/PAGE profile of co-purifying proteins with Flag-Tagged Cas7 expressed in Haloferax cells. Cas8 was detected by MS. ( B ) Reconstitution of physical interaction between purified Methanothermobacter Cas8′ (20 μg) with purified complex of affinity tagged Methanothermobacter Cas5–Cas7 (20 μg). Upper panel shows western blot using anti-(His) 6 antibody to detect (His) 6 Cas8′ and the lower panel used anti-MBP antibody to detect MBP in MBP–Cas5–Cas7. ‘Input’ is a duplicate loading of total amount of used Cas8′ (upper panel) or Cas5–Cas7 (lower panel). Cas8′ was detected in the elution ( E ) after binding to amylose—MBP–Cas5–Cas7 (lane 8) but did not bind to amylose pre-bound with BSA (lane 6). ( C ) Measurements of duplex DNA binding ±PAM by Methanothermobacter Cas5–Cas7 either with or without Cas8′, as labelled. Data values for total DNA binding were calculated for each concentration of Cas5–Cas7 (4, 8, 15 nM) ±Cas8′ (5 nM). ( D ) Corresponding EMSA and western blots for detection of Cas5–Cas7 in a Cas8′ dependent in-gel complex. Lanes 1–5 (left panel) show phosphorimaged EMSA complexes arising from reactions binding of Cas5–Cas7 (complex A) or Cas8′ (B complexes). A new complex C was observed when Cas5–Cas7 and Cas8′ are present. Western blotting detected Cas5–Cas7 in complex C (lanes 8 and 9), as well as complex A (lanes 6, 8 and 9).
    Figure Legend Snippet: Interaction of Cas8 and Cas8′ with Cas5–Cas7 ( A ) Coomassie stained SDS/PAGE profile of co-purifying proteins with Flag-Tagged Cas7 expressed in Haloferax cells. Cas8 was detected by MS. ( B ) Reconstitution of physical interaction between purified Methanothermobacter Cas8′ (20 μg) with purified complex of affinity tagged Methanothermobacter Cas5–Cas7 (20 μg). Upper panel shows western blot using anti-(His) 6 antibody to detect (His) 6 Cas8′ and the lower panel used anti-MBP antibody to detect MBP in MBP–Cas5–Cas7. ‘Input’ is a duplicate loading of total amount of used Cas8′ (upper panel) or Cas5–Cas7 (lower panel). Cas8′ was detected in the elution ( E ) after binding to amylose—MBP–Cas5–Cas7 (lane 8) but did not bind to amylose pre-bound with BSA (lane 6). ( C ) Measurements of duplex DNA binding ±PAM by Methanothermobacter Cas5–Cas7 either with or without Cas8′, as labelled. Data values for total DNA binding were calculated for each concentration of Cas5–Cas7 (4, 8, 15 nM) ±Cas8′ (5 nM). ( D ) Corresponding EMSA and western blots for detection of Cas5–Cas7 in a Cas8′ dependent in-gel complex. Lanes 1–5 (left panel) show phosphorimaged EMSA complexes arising from reactions binding of Cas5–Cas7 (complex A) or Cas8′ (B complexes). A new complex C was observed when Cas5–Cas7 and Cas8′ are present. Western blotting detected Cas5–Cas7 in complex C (lanes 8 and 9), as well as complex A (lanes 6, 8 and 9).

    Techniques Used: Staining, SDS Page, Mass Spectrometry, Purification, Western Blot, Binding Assay, Concentration Assay

    14) Product Images from "NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export"

    Article Title: NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv039

    ( A ) Structures of pCMV128-RLucCTE (test plasmid) and pME-FLuc (control plasmid). ( B ) The test and the control plasmids along with pEGFP or pEGFP-Tap (full-length; wild type or the NTF2L domain mutants) were transfected to 293F cells. A p15-FLAG expression vector was included in the transfections as indicated. At 48 h post transfection, dual-luciferase assay was performed and RLuc/FLuc ratios were calculated. Note that upon overexpression of GFP-Tap and p15-FLAG, expression of RLuc was increased by ∼14-fold as compared with GFP ( P = 1.11 × 10 −7 by Student's t -test). The Tap mutants harboring the alanine-scan mutations in the NTF2L-domain (m1 to m9) activated RLuc expression less efficiently than the wild-type protein ( P -values, m1: 2.55 × 10 −6 , m2: 4.90 × 10 −7 , m3: 1.94 × 10 −7 , m5: 3.06 × 10 −7 , m6: 2.12 × 10 −7 , m7: 4.09 × 10 −6 , m8: 1.09 × 10 −7 , m9: 7.13 × 10 −6 ). ( C ) GFP or GFP-Tap (wild type or the NTF2L-domain mutants) expression plasmids used in (B) were transfected to 293 cells. A p15-FLAG expression vector was included in the transfections as indicated. At 48 h post transfection, total cell extracts were prepared and they were subjected to western blot using anti-GFP (upper panel) and anti-FLAG (lower panel) antibodies.
    Figure Legend Snippet: ( A ) Structures of pCMV128-RLucCTE (test plasmid) and pME-FLuc (control plasmid). ( B ) The test and the control plasmids along with pEGFP or pEGFP-Tap (full-length; wild type or the NTF2L domain mutants) were transfected to 293F cells. A p15-FLAG expression vector was included in the transfections as indicated. At 48 h post transfection, dual-luciferase assay was performed and RLuc/FLuc ratios were calculated. Note that upon overexpression of GFP-Tap and p15-FLAG, expression of RLuc was increased by ∼14-fold as compared with GFP ( P = 1.11 × 10 −7 by Student's t -test). The Tap mutants harboring the alanine-scan mutations in the NTF2L-domain (m1 to m9) activated RLuc expression less efficiently than the wild-type protein ( P -values, m1: 2.55 × 10 −6 , m2: 4.90 × 10 −7 , m3: 1.94 × 10 −7 , m5: 3.06 × 10 −7 , m6: 2.12 × 10 −7 , m7: 4.09 × 10 −6 , m8: 1.09 × 10 −7 , m9: 7.13 × 10 −6 ). ( C ) GFP or GFP-Tap (wild type or the NTF2L-domain mutants) expression plasmids used in (B) were transfected to 293 cells. A p15-FLAG expression vector was included in the transfections as indicated. At 48 h post transfection, total cell extracts were prepared and they were subjected to western blot using anti-GFP (upper panel) and anti-FLAG (lower panel) antibodies.

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Luciferase, Over Expression, Western Blot

    ( A ) Indicated siRNAs were transfected to 293F cells. At 36 h after the siRNA transfection, GFP-fusion vectors encoding wild type and siRNA-resistant Tap (Tap R ) were transfected along with a p15-FLAG expression vector. At 24 h after the second transfection, total cell lysates were prepared and they were subjected to western blot using anti-Tap (upper panel) and anti-β-actin (lower panel) antibodies. Positions of molecular weight markers are shown on the left in kDa. ( B ) 293F cells treated as in (A) were fixed and subjected to in situ hybridization using Cy3-labeled oligo-dT 50 probe. The cells were observed by a confocal microscopy. ( C ) GFP-Tap R fusion vectors harboring point mutations in the NTF2L (m8) and RRM (R 128 K > EE) domains or both (m8+R 128 K > EE) were transfected to 293F cells along with a p15-FLAG expression vector. Total cell extracts prepared at 48 h post transfection were subjected to western blot using anti-GFP (upper panel) and anti-FLAG (lower panel) antibodies. ( D ) Same as in (B), but the GFP-Tap R variants used in (C) were expressed instead of the wild-type protein. ( E ) 293F cells expressing the indicated GFP fusion proteins were irradiated with UV light. Whole cell extracts were prepared and poly (A) + RNA was purified by oligo-dT cellulose chromatography. RNase A-treated whole cell extracts (input) and poly (A) + RNA fractions were analyzed by western blot using anti-GFP (upper panels) and anti-hnRNP C (lower panels) antibodies.
    Figure Legend Snippet: ( A ) Indicated siRNAs were transfected to 293F cells. At 36 h after the siRNA transfection, GFP-fusion vectors encoding wild type and siRNA-resistant Tap (Tap R ) were transfected along with a p15-FLAG expression vector. At 24 h after the second transfection, total cell lysates were prepared and they were subjected to western blot using anti-Tap (upper panel) and anti-β-actin (lower panel) antibodies. Positions of molecular weight markers are shown on the left in kDa. ( B ) 293F cells treated as in (A) were fixed and subjected to in situ hybridization using Cy3-labeled oligo-dT 50 probe. The cells were observed by a confocal microscopy. ( C ) GFP-Tap R fusion vectors harboring point mutations in the NTF2L (m8) and RRM (R 128 K > EE) domains or both (m8+R 128 K > EE) were transfected to 293F cells along with a p15-FLAG expression vector. Total cell extracts prepared at 48 h post transfection were subjected to western blot using anti-GFP (upper panel) and anti-FLAG (lower panel) antibodies. ( D ) Same as in (B), but the GFP-Tap R variants used in (C) were expressed instead of the wild-type protein. ( E ) 293F cells expressing the indicated GFP fusion proteins were irradiated with UV light. Whole cell extracts were prepared and poly (A) + RNA was purified by oligo-dT cellulose chromatography. RNase A-treated whole cell extracts (input) and poly (A) + RNA fractions were analyzed by western blot using anti-GFP (upper panels) and anti-hnRNP C (lower panels) antibodies.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Molecular Weight, In Situ Hybridization, Labeling, Confocal Microscopy, Irradiation, Purification, Chromatography

    15) Product Images from "The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome"

    Article Title: The DNA binding CXC domain of MSL2 is required for faithful targeting the Dosage Compensation Complex to the X chromosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq026

    Recombinant MSL derivatives investigated in this study. ( A ) Schematic representation of MSL2 domain organization and various MSL2 expression constructs. All MSL2 constructs contain a C-terminal FLAG tag. Numbers correspond to the amino acid positions in full-length MSL2. ( B ) Alignment of orthologue CXC domains from the Drosophila melanogaster MSL2 protein (DmCXC) and from the H. sapiens protein KIAA1585 (HsCXC). Black boxes highlight the conserved cysteines ( 37 ). Arrows indicate the introduced point mutations. ( C ) Coomassie-stained SDS–polyacrylamide gel of purified recombinant MSL proteins.
    Figure Legend Snippet: Recombinant MSL derivatives investigated in this study. ( A ) Schematic representation of MSL2 domain organization and various MSL2 expression constructs. All MSL2 constructs contain a C-terminal FLAG tag. Numbers correspond to the amino acid positions in full-length MSL2. ( B ) Alignment of orthologue CXC domains from the Drosophila melanogaster MSL2 protein (DmCXC) and from the H. sapiens protein KIAA1585 (HsCXC). Black boxes highlight the conserved cysteines ( 37 ). Arrows indicate the introduced point mutations. ( C ) Coomassie-stained SDS–polyacrylamide gel of purified recombinant MSL proteins.

    Techniques Used: Recombinant, Expressing, Construct, FLAG-tag, Staining, Purification

    16) Product Images from "Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A-Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase"

    Article Title: Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A-Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01270-12

    S595 phosphorylation is highest in mitotic cells. (A) HeLa cells were transfected with FLAG-eEF2 and synchronized in either early S phase (lane 2) or prometaphase (lanes 3 to 5). The immunoprecipitated eEF2 proteins were subjected to in vitro phosphorylation by cyclin A-CDK2. The top panel shows the amount of eEF2 phosphorylation, and the bottom panel indicates the amount of total eEF2 protein from a representative experiment. eEF2 from nocodazole-arrested mitotic cells was treated with lambda phosphatase prior to the cyclin A-CDK2 reaction represented in lane 4. Cells arrested in mitosis were treated with roscovitine to inhibit mitotic CDKs (lane 5). A, asynchronous; S, hydroxyurea; M, nocodazole; M+Ros, nocodazole plus roscovitine pulse. (B) Combined results of four independent experiments in which the amount of in vitro eEF2 phosphorylation by cyclin A-CDK2 was compared with the amount seen with asynchronous cells. The amount of eEF2 phosphorylation was first normalized to the total amount of eEF2 present in the immunoprecipitates. Error bars indicate the standard deviations of the means of the results of the four independent experiments. Three experiments used hydroxyurea to arrest S-phase cells, whereas the fourth used aphidicolin. (C) Flow cytometric cell cycle profiles of the various cell populations of a representative experiment shown in panel B. (D) Hct116 colon carcinoma cells, 293A cells, or SK-N-AS neuroblastoma cells were synchronized with aphidicolin (S) or nocodazole (M) or grown asynchronously (A). The amounts of total endogenous eEF2 and phospho-T56-eEF2 are shown.
    Figure Legend Snippet: S595 phosphorylation is highest in mitotic cells. (A) HeLa cells were transfected with FLAG-eEF2 and synchronized in either early S phase (lane 2) or prometaphase (lanes 3 to 5). The immunoprecipitated eEF2 proteins were subjected to in vitro phosphorylation by cyclin A-CDK2. The top panel shows the amount of eEF2 phosphorylation, and the bottom panel indicates the amount of total eEF2 protein from a representative experiment. eEF2 from nocodazole-arrested mitotic cells was treated with lambda phosphatase prior to the cyclin A-CDK2 reaction represented in lane 4. Cells arrested in mitosis were treated with roscovitine to inhibit mitotic CDKs (lane 5). A, asynchronous; S, hydroxyurea; M, nocodazole; M+Ros, nocodazole plus roscovitine pulse. (B) Combined results of four independent experiments in which the amount of in vitro eEF2 phosphorylation by cyclin A-CDK2 was compared with the amount seen with asynchronous cells. The amount of eEF2 phosphorylation was first normalized to the total amount of eEF2 present in the immunoprecipitates. Error bars indicate the standard deviations of the means of the results of the four independent experiments. Three experiments used hydroxyurea to arrest S-phase cells, whereas the fourth used aphidicolin. (C) Flow cytometric cell cycle profiles of the various cell populations of a representative experiment shown in panel B. (D) Hct116 colon carcinoma cells, 293A cells, or SK-N-AS neuroblastoma cells were synchronized with aphidicolin (S) or nocodazole (M) or grown asynchronously (A). The amounts of total endogenous eEF2 and phospho-T56-eEF2 are shown.

    Techniques Used: Transfection, Immunoprecipitation, In Vitro, Flow Cytometry

    An intact S595 region is required for efficient T56 phosphorylation. (A) eEF2 T56 phosphorylation in vivo is reduced by the S595A and H599P mutations. The indicated FLAG-eEF2 proteins were immunoprecipitated from transfected 293A cells and immunoblotted with anti-phospho-T56 eEF2 antibody and anti-FLAG. (B) eEF2 S595A is poorly phosphorylated by eEF2K in vitro . FLAG-eEF2 or FLAG-eEF2 S595A eluted from immunoprecipitates was subjected to phosphorylation by eEF2K in vitro , followed by autoradiography and immunoblotting with anti-pT56-eEF2. Input FLAG-eEF2 is shown. (C) S595A and H599P mutations prevent eEF2 phosphorylation by eEF2K in vitro . Immunoprecipitates of the indicated FLAG-eEF2 proteins were phosphorylated in vitro with either cyclin A-CDK2 (first panel) or recombinant eEF2K (fourth panel) or blotted for eEF2 (third panel) and pT56 eEF2 (second panel). eEF2K autophosphorylation is indicated (fourth panel). Panel 5 shows eEF2 and eEF2K abundance in the kinase assay shown in panel 4. (D) eEF2 S595A functions as a translocase. Diphtheria toxin and NAD + were used to inhibit endogenous eEF2 in reticulocyte extracts (lane 2 to 4), and either WT eEF2 (lane 3) or 595A eEF2 (lane 4) was used to reconstitute translation of luciferase protein.
    Figure Legend Snippet: An intact S595 region is required for efficient T56 phosphorylation. (A) eEF2 T56 phosphorylation in vivo is reduced by the S595A and H599P mutations. The indicated FLAG-eEF2 proteins were immunoprecipitated from transfected 293A cells and immunoblotted with anti-phospho-T56 eEF2 antibody and anti-FLAG. (B) eEF2 S595A is poorly phosphorylated by eEF2K in vitro . FLAG-eEF2 or FLAG-eEF2 S595A eluted from immunoprecipitates was subjected to phosphorylation by eEF2K in vitro , followed by autoradiography and immunoblotting with anti-pT56-eEF2. Input FLAG-eEF2 is shown. (C) S595A and H599P mutations prevent eEF2 phosphorylation by eEF2K in vitro . Immunoprecipitates of the indicated FLAG-eEF2 proteins were phosphorylated in vitro with either cyclin A-CDK2 (first panel) or recombinant eEF2K (fourth panel) or blotted for eEF2 (third panel) and pT56 eEF2 (second panel). eEF2K autophosphorylation is indicated (fourth panel). Panel 5 shows eEF2 and eEF2K abundance in the kinase assay shown in panel 4. (D) eEF2 S595A functions as a translocase. Diphtheria toxin and NAD + were used to inhibit endogenous eEF2 in reticulocyte extracts (lane 2 to 4), and either WT eEF2 (lane 3) or 595A eEF2 (lane 4) was used to reconstitute translation of luciferase protein.

    Techniques Used: In Vivo, Immunoprecipitation, Transfection, In Vitro, Autoradiography, Recombinant, Kinase Assay, Luciferase

    eEF2 S595 phosphorylation in vivo . (A) Phosphopeptide maps of eEF2 (map 1) and the indicated mutants (maps 2 to 4) isolated from 293T cells labeled with [ 32 P]orthophosphate. The peptides representing S595 (spots A and B) and T56 (spot C) are indicated. The S595A mutation abrogates the S595 spots in both the WT and H599P backgrounds (maps 2 and 4). Note that the amount of T56 phosphorylation is reduced in the S595A and H599P mutants (maps 2 to 4). Asterisks indicate a minor spot that comigrates with spot A. (B) Amount of overexpression of transfected FLAG-eEF2 relative to endogenous eEF2 (endog-eEF2). Vec, vector. (C) Identification of spot C as containing phosphorylated T56. Cells transfected with WT eEF2 or eEF2 T56A were labeled with orthophosphate and the immunoprecipitated eEF2 proteins analyzed by phosphopeptide mapping. (D) eEF2 phosphorylation is increased by cyclin A-CDK2 expression. U2OS cells were transfected with eEF2 and either cyclin A-CDK2 or empty vector (Vec), and eEF2 was immunoprecipitated after [ 32 P]orthophosphate labeling (top two panels). The bottom panel shows total cyclin A-CDK histone H1 kinase activity. Phosphopeptide mapping reveals increased phosphorylation of S595 (spots A and B) and other sites (arrows). (E) Analysis similar to that described for panel D using eEF2 H599P.
    Figure Legend Snippet: eEF2 S595 phosphorylation in vivo . (A) Phosphopeptide maps of eEF2 (map 1) and the indicated mutants (maps 2 to 4) isolated from 293T cells labeled with [ 32 P]orthophosphate. The peptides representing S595 (spots A and B) and T56 (spot C) are indicated. The S595A mutation abrogates the S595 spots in both the WT and H599P backgrounds (maps 2 and 4). Note that the amount of T56 phosphorylation is reduced in the S595A and H599P mutants (maps 2 to 4). Asterisks indicate a minor spot that comigrates with spot A. (B) Amount of overexpression of transfected FLAG-eEF2 relative to endogenous eEF2 (endog-eEF2). Vec, vector. (C) Identification of spot C as containing phosphorylated T56. Cells transfected with WT eEF2 or eEF2 T56A were labeled with orthophosphate and the immunoprecipitated eEF2 proteins analyzed by phosphopeptide mapping. (D) eEF2 phosphorylation is increased by cyclin A-CDK2 expression. U2OS cells were transfected with eEF2 and either cyclin A-CDK2 or empty vector (Vec), and eEF2 was immunoprecipitated after [ 32 P]orthophosphate labeling (top two panels). The bottom panel shows total cyclin A-CDK histone H1 kinase activity. Phosphopeptide mapping reveals increased phosphorylation of S595 (spots A and B) and other sites (arrows). (E) Analysis similar to that described for panel D using eEF2 H599P.

    Techniques Used: In Vivo, Isolation, Labeling, Mutagenesis, Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Activity Assay

    Cyclin A-CDK2 phosphorylates eEF2 on S595 in vitro . (A) Cyclin A-CDK phosphorylates eEF2. FLAG-eEF2 was eluted from immunoprecipitates (IP) and phosphorylated with myc tag-cyclin A-CDK2 isolated from transfected 293A cells. Top, autoradiograph of phosphorylated eEF2; bottom, eEF2 immunoblot (IB). (B) The six possible eEF2 phosphorylation sites. S435 and S595 contain CDK motifs (underlines). (C) Reduced phosphorylation of eEF2 595A by cyclin A-CDK2. The indicated eEF2 mutants were immunoprecipitated from transfected 293T cells and phosphorylated with recombinant cyclin A-CDK2. Top, autoradiograph of phosphorylated eEF2; bottom, Ponceau stain for input control. (D) FLAG-eEF2 was phosphorylated by recombinant cyclin A-CDK2 (AK2) or cyclin B-CDC2 (BK2). The amount of histone H1 phosphorylation by both enzymes is shown, as well as the FLAG-eEF2 input (IB: anti-FLAG). C, control. (E) eEF2 was phosphorylated with recombinant cyclin A-CDK2 or cyclin E-CDK2 as described for panel D. (F) FLAG-eEF2 was subject to kinase reactions with cyclin A-CDK2 (AK-2), MAPK, or GSK3β as indicated. GSK3β autophosphorylation is noted. (G) GSK3β phosphorylates myc-tagged cyclin E immunoprecipitated from transfected 293A cells. GSK3β autophosphorylation is noted. HC, heavy chain; LC, light chain. (H) MAPK phosphorylates HA–c-Jun immunoprecipitated from transfected 293A cells. (I) MAPK phosphorylates endogenous 4EBP1 immunoprecipitated from 293A cells. MT cyc E, myc-tagged cyclin E.
    Figure Legend Snippet: Cyclin A-CDK2 phosphorylates eEF2 on S595 in vitro . (A) Cyclin A-CDK phosphorylates eEF2. FLAG-eEF2 was eluted from immunoprecipitates (IP) and phosphorylated with myc tag-cyclin A-CDK2 isolated from transfected 293A cells. Top, autoradiograph of phosphorylated eEF2; bottom, eEF2 immunoblot (IB). (B) The six possible eEF2 phosphorylation sites. S435 and S595 contain CDK motifs (underlines). (C) Reduced phosphorylation of eEF2 595A by cyclin A-CDK2. The indicated eEF2 mutants were immunoprecipitated from transfected 293T cells and phosphorylated with recombinant cyclin A-CDK2. Top, autoradiograph of phosphorylated eEF2; bottom, Ponceau stain for input control. (D) FLAG-eEF2 was phosphorylated by recombinant cyclin A-CDK2 (AK2) or cyclin B-CDC2 (BK2). The amount of histone H1 phosphorylation by both enzymes is shown, as well as the FLAG-eEF2 input (IB: anti-FLAG). C, control. (E) eEF2 was phosphorylated with recombinant cyclin A-CDK2 or cyclin E-CDK2 as described for panel D. (F) FLAG-eEF2 was subject to kinase reactions with cyclin A-CDK2 (AK-2), MAPK, or GSK3β as indicated. GSK3β autophosphorylation is noted. (G) GSK3β phosphorylates myc-tagged cyclin E immunoprecipitated from transfected 293A cells. GSK3β autophosphorylation is noted. HC, heavy chain; LC, light chain. (H) MAPK phosphorylates HA–c-Jun immunoprecipitated from transfected 293A cells. (I) MAPK phosphorylates endogenous 4EBP1 immunoprecipitated from 293A cells. MT cyc E, myc-tagged cyclin E.

    Techniques Used: In Vitro, Isolation, Transfection, Autoradiography, Immunoprecipitation, Recombinant, Staining

    17) Product Images from "CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells"

    Article Title: CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24824

    CNPY2 inhibits MYLIP-mediated AR ubiquitination and protein degradation (A) Immunoprecipitation of LNCaP cell extracts with anti-FLAG M2 affinity gel. LNCaP cells were transfected with FLAG-tagged MYLIP expression plasmids for 24 h and used for the immunoprecipitation. AR bound to MYLIP was then detected by immunoblotting. (B) Diagrams representing AR protein structure. K845 (Lys 845) and K847 (Lys847) are the two conserved ubiquitination sites on AR. DBD, DNA binding domain. LBD, Ligand binding domain. (C) Immunoprecipitation of 293T cell extracts with anti-His tag affinity beads. 293T cells were transfected with FLAG-tagged AR (full length, AF-1 or AF-2) expression plasmids and MYLIP-His or His-tag expression plasmids for 24 h and used for the immunoprecipitation. FLAG-ARs bound to MYLIP-His were then detected by immunoblotting with anti-FLAG. (D) MYLIP mediated-ubiquitination of AR was detected by in vivo ubiquitination assay. 293T cells were transfected with FLAG-AR (AF-2, K845R or K847R), MYLIP-His and EGFP-ubiquitin expression plasmids for 24 h and 10 µM MG132 was added to the culture medium 5 h before cell extraction. Cells were lysed and subjected to immunoprecipitation using anti-FLAG M2 affinity gel, followed by immunoblotting with each antibody. (E) In vivo ubiquitination assays were performed using 293T cells transfected with plasmids as indicated. Immunoprecipitation of AR (full length) was done using anti-AR (N-20). (F) In vitro ubiquitination assays were performed using recombinant AR (AF-2)-His, recombinant GST-CNPY2 and immunoprecipitated with FLAG-MYLIP. Reactions were performed with recombinant E1 enzyme, E2 enzyme and ubiquitin at 37° C for 2 h. Ubiquitination of AR was detected by immunoblotting with anti-AR (C-19). (G) Coomassie Brilliant Blue staining with recombinant AR (AF-2)-His protein. (H) Immunoblots using CNPY2 or MYLIP-knockdown LNCaP cell lysates with anti-AR, anti-MYLIP, or anti-CNPY2 antibodies. Band intensity was quantified by Adobe Photoshop. The measurements were normalized to si-Control protein levels that are indicated at the bottom of each band.
    Figure Legend Snippet: CNPY2 inhibits MYLIP-mediated AR ubiquitination and protein degradation (A) Immunoprecipitation of LNCaP cell extracts with anti-FLAG M2 affinity gel. LNCaP cells were transfected with FLAG-tagged MYLIP expression plasmids for 24 h and used for the immunoprecipitation. AR bound to MYLIP was then detected by immunoblotting. (B) Diagrams representing AR protein structure. K845 (Lys 845) and K847 (Lys847) are the two conserved ubiquitination sites on AR. DBD, DNA binding domain. LBD, Ligand binding domain. (C) Immunoprecipitation of 293T cell extracts with anti-His tag affinity beads. 293T cells were transfected with FLAG-tagged AR (full length, AF-1 or AF-2) expression plasmids and MYLIP-His or His-tag expression plasmids for 24 h and used for the immunoprecipitation. FLAG-ARs bound to MYLIP-His were then detected by immunoblotting with anti-FLAG. (D) MYLIP mediated-ubiquitination of AR was detected by in vivo ubiquitination assay. 293T cells were transfected with FLAG-AR (AF-2, K845R or K847R), MYLIP-His and EGFP-ubiquitin expression plasmids for 24 h and 10 µM MG132 was added to the culture medium 5 h before cell extraction. Cells were lysed and subjected to immunoprecipitation using anti-FLAG M2 affinity gel, followed by immunoblotting with each antibody. (E) In vivo ubiquitination assays were performed using 293T cells transfected with plasmids as indicated. Immunoprecipitation of AR (full length) was done using anti-AR (N-20). (F) In vitro ubiquitination assays were performed using recombinant AR (AF-2)-His, recombinant GST-CNPY2 and immunoprecipitated with FLAG-MYLIP. Reactions were performed with recombinant E1 enzyme, E2 enzyme and ubiquitin at 37° C for 2 h. Ubiquitination of AR was detected by immunoblotting with anti-AR (C-19). (G) Coomassie Brilliant Blue staining with recombinant AR (AF-2)-His protein. (H) Immunoblots using CNPY2 or MYLIP-knockdown LNCaP cell lysates with anti-AR, anti-MYLIP, or anti-CNPY2 antibodies. Band intensity was quantified by Adobe Photoshop. The measurements were normalized to si-Control protein levels that are indicated at the bottom of each band.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Binding Assay, Ligand Binding Assay, In Vivo, Ubiquitin Assay, In Vitro, Recombinant, Staining, Western Blot

    18) Product Images from "LRRK2 regulates endoplasmic reticulum–mitochondrial tethering through the PERK‐mediated ubiquitination pathway"

    Article Title: LRRK2 regulates endoplasmic reticulum–mitochondrial tethering through the PERK‐mediated ubiquitination pathway

    Journal: The EMBO Journal

    doi: 10.15252/embj.2018100875

    PERK phosphorylates E3 ubiquitin ligases Peak values of Ca 2+ transients in MEFs of the indicated genotypes treated with LRRK2‐IN‐1 (1 μM). Error bars represent ± SD from six ind ependent experiments. (Upper panel) Diagram showing full‐length PERK tagged with Myc at the N‐terminus and FLAG at the C‐terminus (M‐PERK‐F). The S1P recognition sequence R 33 SLL is mutated to A 33 SLL (M‐PERK(R33A)‐F).(Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against the FLAG epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right. In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase, and [γ‐ 32 P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were probed with antibody against Myc, FLAG, or phosphoserine. [γ‐ 32 P]ATP‐incorporated E3 ubiquitin ligases were visualized by autoradiography (left blot was exposed for 24 h, and right blot for 36 h). In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA‐tagged Ubl, and His‐tagged mitofusin 2 in the presence of E1 enzyme, UbcH7, and ATP. E3 ubiquitin ligases were initially phosphorylated by PERK or PERK(K618R) in vitro . Phosphorylated E3 ubiquitin ligases were subjected to in vitro ubiquitination. Mitofusin 2 precipitated with Ni‐NTA was subjected to SDS/PAGE. Blots were probed with antibody against HA or mitofusin 2. Mfn2: mitofusin 2, Ub: ubiquitin. Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)‐expressing MEFs transfected with the increasing amounts of LRRK2‐d1‐V5 (1, 5, 25 μg/10 6 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN, and Parkin were immunoprecipitated with the corresponding antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti‐mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN, and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ± SD from four independent experiments. Data information: For graphs (A and E), the P values were determined by a Mann–Whitney U ‐test. ns = not significant, * P
    Figure Legend Snippet: PERK phosphorylates E3 ubiquitin ligases Peak values of Ca 2+ transients in MEFs of the indicated genotypes treated with LRRK2‐IN‐1 (1 μM). Error bars represent ± SD from six ind ependent experiments. (Upper panel) Diagram showing full‐length PERK tagged with Myc at the N‐terminus and FLAG at the C‐terminus (M‐PERK‐F). The S1P recognition sequence R 33 SLL is mutated to A 33 SLL (M‐PERK(R33A)‐F).(Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against the FLAG epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right. In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase, and [γ‐ 32 P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were probed with antibody against Myc, FLAG, or phosphoserine. [γ‐ 32 P]ATP‐incorporated E3 ubiquitin ligases were visualized by autoradiography (left blot was exposed for 24 h, and right blot for 36 h). In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA‐tagged Ubl, and His‐tagged mitofusin 2 in the presence of E1 enzyme, UbcH7, and ATP. E3 ubiquitin ligases were initially phosphorylated by PERK or PERK(K618R) in vitro . Phosphorylated E3 ubiquitin ligases were subjected to in vitro ubiquitination. Mitofusin 2 precipitated with Ni‐NTA was subjected to SDS/PAGE. Blots were probed with antibody against HA or mitofusin 2. Mfn2: mitofusin 2, Ub: ubiquitin. Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)‐expressing MEFs transfected with the increasing amounts of LRRK2‐d1‐V5 (1, 5, 25 μg/10 6 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN, and Parkin were immunoprecipitated with the corresponding antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti‐mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN, and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ± SD from four independent experiments. Data information: For graphs (A and E), the P values were determined by a Mann–Whitney U ‐test. ns = not significant, * P

    Techniques Used: Sequencing, Transfection, Immunoprecipitation, FLAG-tag, SDS Page, Staining, In Vitro, Kinase Assay, Isolation, Autoradiography, Ubiquitin Assay, Expressing, MANN-WHITNEY

    Amino acid sequence of mouse PERK in the region of the S1P cleavage site Diagram showing full‐length PERK. TM, transmembrane domain; S/T kinase, serine/threonine kinase. Amino acid sequences of PERK that are necessary for S1P cleavage are highlighted in red. The box indicates the position of the transmembrane domain. Diagram showing full‐length PERK tagged with Myc at the N‐terminus and FLAG at the C‐terminus (M‐PERK‐F). The S1P recognition sequence R 33 SLL is mutated to A 33 SLL (M‐PERK(R33A)‐F). Under ER stress, Myc‐PERK‐FLAG is cleaved by S1P at L 36 ‐A 37 and released into the cytosol; the soluble domain is detected by antibody against FLAG, but not Myc.
    Figure Legend Snippet: Amino acid sequence of mouse PERK in the region of the S1P cleavage site Diagram showing full‐length PERK. TM, transmembrane domain; S/T kinase, serine/threonine kinase. Amino acid sequences of PERK that are necessary for S1P cleavage are highlighted in red. The box indicates the position of the transmembrane domain. Diagram showing full‐length PERK tagged with Myc at the N‐terminus and FLAG at the C‐terminus (M‐PERK‐F). The S1P recognition sequence R 33 SLL is mutated to A 33 SLL (M‐PERK(R33A)‐F). Under ER stress, Myc‐PERK‐FLAG is cleaved by S1P at L 36 ‐A 37 and released into the cytosol; the soluble domain is detected by antibody against FLAG, but not Myc.

    Techniques Used: Sequencing

    19) Product Images from "O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β *"

    Article Title: O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.751420

    OEC has elongation factor properties. A , purification of OEC. Shown is a silver-stained PAGE of FLAG-tagged SPT5/OEC as well as input and flow-through of the M2-agarose resin used to capture the FLAG epitope. B , OEC contains OGA, SPT5, and TIF1β.
    Figure Legend Snippet: OEC has elongation factor properties. A , purification of OEC. Shown is a silver-stained PAGE of FLAG-tagged SPT5/OEC as well as input and flow-through of the M2-agarose resin used to capture the FLAG epitope. B , OEC contains OGA, SPT5, and TIF1β.

    Techniques Used: Purification, Staining, Polyacrylamide Gel Electrophoresis, Flow Cytometry, FLAG-tag

    20) Product Images from "The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase"

    Article Title: The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0140755

    Thermal stability assays and partial proteolysis mapping of ChlR1 proteins. ( A ) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. ( B ) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.
    Figure Legend Snippet: Thermal stability assays and partial proteolysis mapping of ChlR1 proteins. ( A ) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. ( B ) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.

    Techniques Used: Purification, Concentration Assay, SDS Page, Western Blot

    ATP hydrolysis and ATP binding assays of ChlR1 proteins. ( A ) A representative image of ChlR1 ATP hydrolysis detected by TLC. ( B ) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. ( C ) ATP binding by wild-type ChlR1 and mutant protein. α 32 P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. ( D ) A representative image of filter dot blot assays of ChlR1 proteins binding α 32 P-ATP. ( E ) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.
    Figure Legend Snippet: ATP hydrolysis and ATP binding assays of ChlR1 proteins. ( A ) A representative image of ChlR1 ATP hydrolysis detected by TLC. ( B ) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. ( C ) ATP binding by wild-type ChlR1 and mutant protein. α 32 P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. ( D ) A representative image of filter dot blot assays of ChlR1 proteins binding α 32 P-ATP. ( E ) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.

    Techniques Used: Binding Assay, Thin Layer Chromatography, Western Blot, Mutagenesis, Filtration, Chromatography, Dot Blot

    Determination of ChlR1 protein oligomerization state. ( A ) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. ( B ) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. ( C ) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. ( D ) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. ( E ) The fractions in D were immunoblotted with an anti-FLAG antibody. ( F ) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.
    Figure Legend Snippet: Determination of ChlR1 protein oligomerization state. ( A ) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. ( B ) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. ( C ) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. ( D ) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. ( E ) The fractions in D were immunoblotted with an anti-FLAG antibody. ( F ) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.

    Techniques Used: Staining, SDS Page, Molecular Weight, Size-exclusion Chromatography, Helicase Assay

    Purification and identification of ChlR1 proteins. ( A ) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. ( B-C ) Western-blot analysis of the purified proteins with antibodies ChlR1 ( B ) and FLAG ( C ).
    Figure Legend Snippet: Purification and identification of ChlR1 proteins. ( A ) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. ( B-C ) Western-blot analysis of the purified proteins with antibodies ChlR1 ( B ) and FLAG ( C ).

    Techniques Used: Purification, Staining, Western Blot

    21) Product Images from "In Vitro Reconstitution of Yeast tUTP/UTP A and UTP B Subcomplexes Provides New Insights into Their Modular Architecture"

    Article Title: In Vitro Reconstitution of Yeast tUTP/UTP A and UTP B Subcomplexes Provides New Insights into Their Modular Architecture

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114898

    Yeast tUTP subcomplex reconstitution in insect cells. All candidate tUTP components were co-expressed in SF21 insect cells infected with baculoviruses containing bacmid K2000. Proteins identified by MS analysis are indicated as Nan1, ▪; Utp10, □; Utp4, ▴; Utp5, ♦; Utp8, •; Utp9, ○ and Utp15, ◊. ( A ) Two-step affinity purification using two different bait proteins. Lysates of 2×10 8 infected cells were used in the first affinity purification step to purify Utp15-FLAG-containing component with anti-FLAG affinity matrix which were eluted with the FLAG peptide (Lane 1). 90% of the eluted material was used for the second affinity purification step with anti-HA affinity matrix to purify Nan1-HA containing components, which were eluted with the HA peptide (Lane 2). The composition of both eluates was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. ( B ) Lysates of 8×10 7 SF21 cells infected with baculovirus K2000 were cleared by low-speed centrifugation as described (N samples) and half of the sample was further cleared by ultracentrifugation (200000× g , 1 h, 4°C, U samples). Utp15-FLAG-containing components were purified from both lysates using anti-FLAG affinity matrix and eluted with the FLAG peptide. The eluted material (10%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. ( C ) Utp15-FLAG-containing components were purified from lysates of 4×10 7 infected cells using anti-FLAG affinity matrix and eluted with the FLAG peptide. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), the eluate (E, 10%) and the fractions (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and by WB using antibodies against HA (middle panel) or FLAG (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification of the corresponding protein by MS analysis is indicated.
    Figure Legend Snippet: Yeast tUTP subcomplex reconstitution in insect cells. All candidate tUTP components were co-expressed in SF21 insect cells infected with baculoviruses containing bacmid K2000. Proteins identified by MS analysis are indicated as Nan1, ▪; Utp10, □; Utp4, ▴; Utp5, ♦; Utp8, •; Utp9, ○ and Utp15, ◊. ( A ) Two-step affinity purification using two different bait proteins. Lysates of 2×10 8 infected cells were used in the first affinity purification step to purify Utp15-FLAG-containing component with anti-FLAG affinity matrix which were eluted with the FLAG peptide (Lane 1). 90% of the eluted material was used for the second affinity purification step with anti-HA affinity matrix to purify Nan1-HA containing components, which were eluted with the HA peptide (Lane 2). The composition of both eluates was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. ( B ) Lysates of 8×10 7 SF21 cells infected with baculovirus K2000 were cleared by low-speed centrifugation as described (N samples) and half of the sample was further cleared by ultracentrifugation (200000× g , 1 h, 4°C, U samples). Utp15-FLAG-containing components were purified from both lysates using anti-FLAG affinity matrix and eluted with the FLAG peptide. The eluted material (10%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. ( C ) Utp15-FLAG-containing components were purified from lysates of 4×10 7 infected cells using anti-FLAG affinity matrix and eluted with the FLAG peptide. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), the eluate (E, 10%) and the fractions (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and by WB using antibodies against HA (middle panel) or FLAG (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification of the corresponding protein by MS analysis is indicated.

    Techniques Used: Infection, Mass Spectrometry, Affinity Purification, SDS Page, Staining, Centrifugation, Purification, Filtration, Western Blot, Marker

    Yeast UTP B subcomplex reconstitution in insect cells. All selected UTP B components were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991 or K1992. The protein content of the indicated bands was identified by MS and are indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. ( A ) Lysates of 2×10 8 cells infected with K1991were used for two-step affinity purification. Pwp2-TAP was used as the bait protein in the first affinity purification step with IgG-coupled Sepharose resin, and Pwp2-containing components were eluted with TEV protease (Lane 1). Utp6-HA-containing components were purified from 90% of the first elution sample using anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of the eluate (5% each) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. ( B ) Lysates of 2×10 8 cells infected with K1992 were used for two-step affinity purification. Utp12-FLAG was purified with anti-FLAG affinity matrix and eluted with the FLAG peptide during the first affinity purification step (Lane 1). A 90% aliquot of the eluted material was used to purify Utp6-HA-containing components with anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of both eluates (5%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. ( C ) Lysates of 8×10 7 SF21 cells infected with bacmid K1991 were cleared by the low-speed centrifugation described in the normal protocol (N samples), and half was further cleared by ultracentrifugation (200000× g , 1 h, 4°C, U samples). Pwp2-TAP-containing components were purified from both lysates using IgG-coupled Sepharose resin and eluted with TEV protease. A 10% aliquot of the eluted material was analyzed on a 4-12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed with MS. ( D ) Pwp2-TAP-containing components were purified from lysates of 4×10 7 infected cells (K1991) using IgG-coupled Sepharose resin and TEV elution. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), flow through from the first purification (FT, 0,03%), the eluate from the affinity column (E, 10%), and the fractions from the gel filtration column (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and WB with antibodies against CBP (middle panel) or HA (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification by MS analysis of the corresponding protein is indicated.
    Figure Legend Snippet: Yeast UTP B subcomplex reconstitution in insect cells. All selected UTP B components were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991 or K1992. The protein content of the indicated bands was identified by MS and are indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. ( A ) Lysates of 2×10 8 cells infected with K1991were used for two-step affinity purification. Pwp2-TAP was used as the bait protein in the first affinity purification step with IgG-coupled Sepharose resin, and Pwp2-containing components were eluted with TEV protease (Lane 1). Utp6-HA-containing components were purified from 90% of the first elution sample using anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of the eluate (5% each) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. ( B ) Lysates of 2×10 8 cells infected with K1992 were used for two-step affinity purification. Utp12-FLAG was purified with anti-FLAG affinity matrix and eluted with the FLAG peptide during the first affinity purification step (Lane 1). A 90% aliquot of the eluted material was used to purify Utp6-HA-containing components with anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of both eluates (5%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. ( C ) Lysates of 8×10 7 SF21 cells infected with bacmid K1991 were cleared by the low-speed centrifugation described in the normal protocol (N samples), and half was further cleared by ultracentrifugation (200000× g , 1 h, 4°C, U samples). Pwp2-TAP-containing components were purified from both lysates using IgG-coupled Sepharose resin and eluted with TEV protease. A 10% aliquot of the eluted material was analyzed on a 4-12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed with MS. ( D ) Pwp2-TAP-containing components were purified from lysates of 4×10 7 infected cells (K1991) using IgG-coupled Sepharose resin and TEV elution. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), flow through from the first purification (FT, 0,03%), the eluate from the affinity column (E, 10%), and the fractions from the gel filtration column (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and WB with antibodies against CBP (middle panel) or HA (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification by MS analysis of the corresponding protein is indicated.

    Techniques Used: Infection, Mass Spectrometry, Affinity Purification, Purification, SDS Page, Staining, Centrifugation, Filtration, Flow Cytometry, Affinity Column, Western Blot, Marker

    22) Product Images from "PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis"

    Article Title: PI3K/AKT activation induces PTEN ubiquitination and destabilization accelerating tumourigenesis

    Journal: Nature Communications

    doi: 10.1038/ncomms8769

    Active AKT induces the stabilization of MKRN1. ( a ) Overexpression of HA-tagged Myr-AKT but not K179M induces increased levels of endogenous MKRN1 or ectopically expressed FLAG-MKRN1 in H1299 cells. ( b ) Myr-AKT stabilizes endogenous MKRN1. H1299 cells were transfected with the indicated plasmid for 24 h and then treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) The half-life of the endogenous MKRN1 protein was determined in EGF (100 ng ml −1 )-stimulated ME-180 cells. ( b,c ) The amount of MKRN1 was determined using western blotting after normalization to actin. (bottom panel, data shown are means±s.d.; n =3). ( d ) H1299 cells transfected with the indicated plasmid were treated with 10 μM MG132 or LLnL for 4 h. ( e ) EGF-dependent MKRN1 ubiquitination is reduced by AKT ablation. ME-180 cells transduced with siAKT1 were treated with EGF (100 ng ml −1 ), followed by MG132 (10 μM) for 4 h. The lysates were immunoprecipitated using an anti-MKRN1 antibody, followed by immunoblotting with an HRP-conjugated anti-Ub antibody under denaturing conditions. ( f ) The protein half-life of the S109D mutant is longer than that of the WT protein. H1299 cells were transfected with FLAG-MKRN1 WT or S109D and then treated with CHX (100 μg ml −1 ) for the indicated time points. Bottom panel: the graphs indicate the relative amounts of MKRN1 protein compared with the levels of actin in the western blot (data shown are means±s.d.; n =3). ( g ) Ubiquitination status of the S109D mutant. H1299 cells were transfected with the indicated plasmids and then treated with MG132 (10 μM). Cells were lysed in 6 M guanidine-HCl, and ubiquitinated proteins were purified using Ni 2+ -NTA beads. His-purified proteins were detected by immunoblotting.
    Figure Legend Snippet: Active AKT induces the stabilization of MKRN1. ( a ) Overexpression of HA-tagged Myr-AKT but not K179M induces increased levels of endogenous MKRN1 or ectopically expressed FLAG-MKRN1 in H1299 cells. ( b ) Myr-AKT stabilizes endogenous MKRN1. H1299 cells were transfected with the indicated plasmid for 24 h and then treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) The half-life of the endogenous MKRN1 protein was determined in EGF (100 ng ml −1 )-stimulated ME-180 cells. ( b,c ) The amount of MKRN1 was determined using western blotting after normalization to actin. (bottom panel, data shown are means±s.d.; n =3). ( d ) H1299 cells transfected with the indicated plasmid were treated with 10 μM MG132 or LLnL for 4 h. ( e ) EGF-dependent MKRN1 ubiquitination is reduced by AKT ablation. ME-180 cells transduced with siAKT1 were treated with EGF (100 ng ml −1 ), followed by MG132 (10 μM) for 4 h. The lysates were immunoprecipitated using an anti-MKRN1 antibody, followed by immunoblotting with an HRP-conjugated anti-Ub antibody under denaturing conditions. ( f ) The protein half-life of the S109D mutant is longer than that of the WT protein. H1299 cells were transfected with FLAG-MKRN1 WT or S109D and then treated with CHX (100 μg ml −1 ) for the indicated time points. Bottom panel: the graphs indicate the relative amounts of MKRN1 protein compared with the levels of actin in the western blot (data shown are means±s.d.; n =3). ( g ) Ubiquitination status of the S109D mutant. H1299 cells were transfected with the indicated plasmids and then treated with MG132 (10 μM). Cells were lysed in 6 M guanidine-HCl, and ubiquitinated proteins were purified using Ni 2+ -NTA beads. His-purified proteins were detected by immunoblotting.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Western Blot, Transduction, Immunoprecipitation, Mutagenesis, Purification

    AKT phosphorylates serine 109 of MKRN1. ( a,b ) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. ( c ) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. ( d ) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml −1 ), and MKRN1-bound endogenous AKT1 was immunoblotted. ( e ) A consensus AKT phosphorylation site is present in MKRN1. ( f ) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ- 32 P]ATP, and 32 P incorporation was detected by autoradiography. ( g ) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. ( h ) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml −1 ) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. ( i ) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml −1 ). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.
    Figure Legend Snippet: AKT phosphorylates serine 109 of MKRN1. ( a,b ) The interaction between ectopically expressed MKRN1 and AKT1 was demonstrated using a co-immunoprecipitation assay. ( c ) A GST pull-down assay revealed the direct interaction between MKRN1 and AKT1. GST-MKRN1 purified from bacteria and in vitro translated HA-AKT1 were incubated under cell-free conditions, and GST-MKRN1 was pulled down by glutathione Sepharose beads. ( d ) Endogenous MKRN1 was immunoprecipitated from ME-180 cells with or without EGF treatment (100 ng ml −1 ), and MKRN1-bound endogenous AKT1 was immunoblotted. ( e ) A consensus AKT phosphorylation site is present in MKRN1. ( f ) AKT directly phosphorylates MKRN1 WT but not the S109A mutant. An in vitro phosphorylation assay was performed using bacterially produced GST-AKT1 and FLAG-MKRN1 (WT or S109A), which was purified from FLAG-MKRN1-transfected HEK293T cells. Purified proteins were incubated with [γ- 32 P]ATP, and 32 P incorporation was detected by autoradiography. ( g ) Constitutively active, but not inactive, AKT phosphorylates MKRN1 on serine 109. H1299 cells were co-transfected with FLAG-MKRN1 (WT or S109A) and HA-tagged Myr-AKT or K179M. Phosphorylation of ectopically expressed MKRN1 was detected by an in vivo phosphorylation assay (IP panel); WCL was also analysed. ( h ) AKT phosphorylates MKRN1 upon EGF treatment. After serum starvation, ME-180 cells were stimulated by EGF (100 ng ml −1 ) in the absence or presence of MG132 (10 μM) for 4 h and were examined in an in vivo phosphorylation assay. Endogenous phospho-MKRN1 was immunoprecipitated using an anti-phosphoserine antibody (IP panel), and WCL was immunoblotted. ( i ) EGF-induced MKRN1 phosphorylation is inhibited by AKT ablation. ME-180 cells were transduced with control siRNA or AKT1 siRNA (siAKT1) and subsequently serum starved, followed by treatment with EGF (100 ng ml −1 ). Cell extracts were analysed using an in vivo phosphorylation assay. Endogenous phospho-MKRN1 is indicated in the IP panel.

    Techniques Used: Co-Immunoprecipitation Assay, Pull Down Assay, Purification, In Vitro, Incubation, Immunoprecipitation, Mutagenesis, Phosphorylation Assay, Produced, Transfection, Autoradiography, In Vivo, Transduction

    MKRN1 induces the ubiquitination and degradation of PTEN. ( a ) MKRN1 degrades both ectopically expressed PTEN and endogenous PTEN. H1299 cells were transfected with the indicated plasmid. GFP was used as the transfection control. ( b ) MKRN1 overexpression decreases endogenous PTEN stability. H1299 cells were transfected with the MKRN1 expressing plasmid for 24 h and then were treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) MKRN1 RNAi stabilizes PTEN. ME-180 cells were transduced with siControl or siMKRN1 #7, followed by the addition of CHX at the indicated time points. ( b,c ) The graph represents the values obtained after densitometry analysis. The percentage of the remaining PTEN protein after CHX addition is plotted (bottom panel, data shown are means±s.d.; n =3). ( d ) MKRN1 induces PTEN ubiquitination. To identify PTEN ubiquitination, H1299 cells were transfected with HA-Ub and the indicated plasmids, followed by treatment with MG132 (10 μM) for 6 h. HA-tagged ubiquitinated PTEN was purified by immunoprecipitation using an anti-FLAG antibody in 1% SDS buffer, followed by immunoblotting using an anti-HA antibody. ( e ) In vitro ubiquitination of PTEN via MKRN1. FLAG-PTEN proteins obtained from HEK293T cells using anti-FLAG beads were incubated with E1, E2 and ubiquitin (Ub) in the absence or presence of ATP along with GST, GST-MKRN1 or H307E (bacterially purified), as indicated for the in vitro ubiquitination of PTEN. ( f ) The ubiquitination status of endogenous PTEN upon MKRN1 or AKT1 ablation. Lysates from MKRN1- or AKT1-knockdown and MG132 (10 μM)-treated ME-180 cells were immunoprecipitated with an anti-PTEN antibody, and ubiquitinated PTEN was then immunoblotted using an HRP-conjugated anti-Ub antibody under denaturing conditions.
    Figure Legend Snippet: MKRN1 induces the ubiquitination and degradation of PTEN. ( a ) MKRN1 degrades both ectopically expressed PTEN and endogenous PTEN. H1299 cells were transfected with the indicated plasmid. GFP was used as the transfection control. ( b ) MKRN1 overexpression decreases endogenous PTEN stability. H1299 cells were transfected with the MKRN1 expressing plasmid for 24 h and then were treated with CHX (100 μg ml −1 ) at the indicated time points. ( c ) MKRN1 RNAi stabilizes PTEN. ME-180 cells were transduced with siControl or siMKRN1 #7, followed by the addition of CHX at the indicated time points. ( b,c ) The graph represents the values obtained after densitometry analysis. The percentage of the remaining PTEN protein after CHX addition is plotted (bottom panel, data shown are means±s.d.; n =3). ( d ) MKRN1 induces PTEN ubiquitination. To identify PTEN ubiquitination, H1299 cells were transfected with HA-Ub and the indicated plasmids, followed by treatment with MG132 (10 μM) for 6 h. HA-tagged ubiquitinated PTEN was purified by immunoprecipitation using an anti-FLAG antibody in 1% SDS buffer, followed by immunoblotting using an anti-HA antibody. ( e ) In vitro ubiquitination of PTEN via MKRN1. FLAG-PTEN proteins obtained from HEK293T cells using anti-FLAG beads were incubated with E1, E2 and ubiquitin (Ub) in the absence or presence of ATP along with GST, GST-MKRN1 or H307E (bacterially purified), as indicated for the in vitro ubiquitination of PTEN. ( f ) The ubiquitination status of endogenous PTEN upon MKRN1 or AKT1 ablation. Lysates from MKRN1- or AKT1-knockdown and MG132 (10 μM)-treated ME-180 cells were immunoprecipitated with an anti-PTEN antibody, and ubiquitinated PTEN was then immunoblotted using an HRP-conjugated anti-Ub antibody under denaturing conditions.

    Techniques Used: Transfection, Plasmid Preparation, Over Expression, Expressing, Transduction, Purification, Immunoprecipitation, In Vitro, Incubation

    23) Product Images from "The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage"

    Article Title: The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36235-0

    UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E , F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G , H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) were transfected with the indicated siRNAs and subjected to clonogenic survival assay after IR (Mean ± SEM, n = 3). *p
    Figure Legend Snippet: UCHL3 facilitates classical NHEJ. (A) Schematic representation of a fluorescent reporter assay measuring c-NHEJ efficiency. I-SceI digestion sites and inserted stop codon are indicated by arrow heads and red characters, respectively. (B) U2OS cells transfected with the indicated siRNAs were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (C) U2OS or UCHL3 KO#2 cells transfected with the indicated plasmid coding FLAG (empty vector: EV) or FLAG-UCHL3 were subjected to c-NHEJ assay. The efficiency of c-NHEJ was normalized to EV transfected U2OS cells and set to 100% (Mean ± SEM, n = 3). (D) U2OS cells transfected with the indicated siRNAs were subjected to direct-repeat GFP assay. The efficiency of homology mediated repair was normalized to control siRNA-transfected cells and set to 100% (Mean ± SEM, n = 3). (E , F) U2OS (WT) or UCHL3 KO#1 cells transfected with the indicated siRNAs were processed for immunoblotting analysis (E) or subjected to clonogenic survival assay after IR (F) (Mean ± SEM, n = 3). (G , H) U2OS cells stably expressing FLAG-UCHL3 (wild-type: WT) (G) or catalytically inactive mutant (C95A) (H) were transfected with the indicated siRNAs and subjected to clonogenic survival assay after IR (Mean ± SEM, n = 3). *p

    Techniques Used: Non-Homologous End Joining, Reporter Assay, Transfection, Plasmid Preparation, Clonogenic Cell Survival Assay, Stable Transfection, Expressing, Mutagenesis

    UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability. (A) U2OS cells transfected with a plasmid expressing FLAG-UCHL3 were treated with phleomycin. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (B) U2OS cells transfected with a plasmid expressing either FLAG-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) were treated with phleomycin or mock treated. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (C) U2OS cells transfected with a plasmid expressing either FLAG or FLAG-UCHL3 were treated with phleomycin for 1 hour and further cultured for the indicated time periods after removal of phleomycin. Cells were processed for immunoprecipitation with anti-FLAG antibody followed by immunoblotting analyses with the indicated antibodies. (D) U2OS cells transfected with the indicated siRNAs were further transfected with a plasmid expressing either GFP-UCHL3 or GFP. Following phleomycin treatment, cell extracts were subjected to immunoprecipitation with an anti-GFP antibody and ensuing immunoblotting analysis with the indicated antibodies. The plasmid expressing GFP was used as a negative control. (E) U2OS cells stably expressing FLAG-UCHL3 (WT, C95A, S75A or S75E) were transfected with siRNA targeting endogenous UCHL3. Cells were incubated with cycloheximide (100 μg/ml) for 1 hour prior to IR (10 Gy) and cultured for the indicated time periods after IR. Immunoblotting analyses were performed with the indicated antibodies. For anti-FLAG antibody detection, short exposure (short exp.) and long exposure (long exp.) of films are shown. Full-length blots are presented in Supplementary Fig. S12 .
    Figure Legend Snippet: UCHL3 phosphorylation requiring its catalytic activity and downstream NHEJ factors regulates UCHL3 stability. (A) U2OS cells transfected with a plasmid expressing FLAG-UCHL3 were treated with phleomycin. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (B) U2OS cells transfected with a plasmid expressing either FLAG-UCHL3 (wild-type: WT) or catalytically inactive mutant (C95A) were treated with phleomycin or mock treated. Immunoprecipitation with an anti-FLAG antibody was carried out, followed by immunoblotting analysis with the indicated antibodies. Transfection with the plasmid coding FLAG was used as a negative control. (C) U2OS cells transfected with a plasmid expressing either FLAG or FLAG-UCHL3 were treated with phleomycin for 1 hour and further cultured for the indicated time periods after removal of phleomycin. Cells were processed for immunoprecipitation with anti-FLAG antibody followed by immunoblotting analyses with the indicated antibodies. (D) U2OS cells transfected with the indicated siRNAs were further transfected with a plasmid expressing either GFP-UCHL3 or GFP. Following phleomycin treatment, cell extracts were subjected to immunoprecipitation with an anti-GFP antibody and ensuing immunoblotting analysis with the indicated antibodies. The plasmid expressing GFP was used as a negative control. (E) U2OS cells stably expressing FLAG-UCHL3 (WT, C95A, S75A or S75E) were transfected with siRNA targeting endogenous UCHL3. Cells were incubated with cycloheximide (100 μg/ml) for 1 hour prior to IR (10 Gy) and cultured for the indicated time periods after IR. Immunoblotting analyses were performed with the indicated antibodies. For anti-FLAG antibody detection, short exposure (short exp.) and long exposure (long exp.) of films are shown. Full-length blots are presented in Supplementary Fig. S12 .

    Techniques Used: Activity Assay, Non-Homologous End Joining, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Negative Control, Mutagenesis, Cell Culture, Stable Transfection, Incubation

    24) Product Images from "Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae"

    Article Title: Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn925

    Rex1p displays tRNA 3′end processing activity in vivo and in vitro . ( A ) A diagram of Rex1p showing the conserved amino acids found in the three Exo motifs. An alignment of Rex1, 2, 3 and 4, and E. coli RNase T protein sequence is shown underneath to illustrate the conservation of the D × E sequence found in the ExoI domain. ( B ) Wt and mutant FLAG-tagged Rex1p were purified from yeast (strains Y438 and Y495, respectively) using affinity chromatography. Purified protein was subjected to SDS-PAGE and visualized by Coomassie staining. The positions of molecular weight standards (Broad Range Protein Marker, New England Bioloabs) are indicated. ( C ) Gel purified 32 P- 5′ end labeled tRNAs i Met (∼ 10 pM) were incubated in buffer alone or with Wt or mutant Rex1p (∼7.5 nM) at 30°C for 10 min. Reaction products were separated on a 10% denaturing polyacrylamide gel and visualized by autoradiography. The positions of RNAs of known length (Decade Marker, Ambion) are indicated (in nucleotides). ( D ) Wt Rex1p (∼7.5 nM) was incubated with labeled ‘ IMT3 ’ tRNA i Met (∼20 pM) for known periods of time from 0.5–7 min, as indicated. Control reactions with ‘ IMT3 ’ tRNA i Met lacking either enzyme or Mg 2+ are shown, as well as a control reaction lacking enzyme that contained the ‘mature’ tRNA i Met . After separation on a denaturing 10% polyacrylamide gel, results were visualized using phosphorimaging. ( E ) Northern analysis of total RNA (10 μg) isolated from a trm6-504 rex1Δ strain carrying an empty vector (pRS316), or a plasmid with galactose-inducible Wt REX1 (pAV101) or mutant rex1 (p532), grown under non-inducing or inducing conditions. The blot was probed with a radiolabeled oligonucleotide complementary to tRNA i Met (JA11) and the results visualized using autoradiography.
    Figure Legend Snippet: Rex1p displays tRNA 3′end processing activity in vivo and in vitro . ( A ) A diagram of Rex1p showing the conserved amino acids found in the three Exo motifs. An alignment of Rex1, 2, 3 and 4, and E. coli RNase T protein sequence is shown underneath to illustrate the conservation of the D × E sequence found in the ExoI domain. ( B ) Wt and mutant FLAG-tagged Rex1p were purified from yeast (strains Y438 and Y495, respectively) using affinity chromatography. Purified protein was subjected to SDS-PAGE and visualized by Coomassie staining. The positions of molecular weight standards (Broad Range Protein Marker, New England Bioloabs) are indicated. ( C ) Gel purified 32 P- 5′ end labeled tRNAs i Met (∼ 10 pM) were incubated in buffer alone or with Wt or mutant Rex1p (∼7.5 nM) at 30°C for 10 min. Reaction products were separated on a 10% denaturing polyacrylamide gel and visualized by autoradiography. The positions of RNAs of known length (Decade Marker, Ambion) are indicated (in nucleotides). ( D ) Wt Rex1p (∼7.5 nM) was incubated with labeled ‘ IMT3 ’ tRNA i Met (∼20 pM) for known periods of time from 0.5–7 min, as indicated. Control reactions with ‘ IMT3 ’ tRNA i Met lacking either enzyme or Mg 2+ are shown, as well as a control reaction lacking enzyme that contained the ‘mature’ tRNA i Met . After separation on a denaturing 10% polyacrylamide gel, results were visualized using phosphorimaging. ( E ) Northern analysis of total RNA (10 μg) isolated from a trm6-504 rex1Δ strain carrying an empty vector (pRS316), or a plasmid with galactose-inducible Wt REX1 (pAV101) or mutant rex1 (p532), grown under non-inducing or inducing conditions. The blot was probed with a radiolabeled oligonucleotide complementary to tRNA i Met (JA11) and the results visualized using autoradiography.

    Techniques Used: Activity Assay, In Vivo, In Vitro, Sequencing, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Staining, Molecular Weight, Marker, Labeling, Incubation, Autoradiography, Northern Blot, Isolation, Plasmid Preparation

    25) Product Images from "Adducin‐1 is essential for spindle pole integrity through its interaction with TPX2"

    Article Title: Adducin‐1 is essential for spindle pole integrity through its interaction with TPX2

    Journal: EMBO Reports

    doi: 10.15252/embr.201745607

    Supernumerary centrosomes induced by ADD1 depletion result from centriole splitting during mitosis HeLa cells were infected with lentiviruses expressing shRNAs specific to ADD1 (sh‐ADD1), Aurora‐B (sh‐AurB), or luciferase (sh‐Luc.). FLAG‐ADD1 WT or mutants (S726A and S726D) were re‐expressed into the cells whose endogenous ADD1 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. The cells were stained for γ‐tubulin (red), α‐tubulin (green), and DNA (blue). Scale bars, 5 μm. The percentage of multiple γ‐tubulin foci in the total counted mitotic cells was measured (302–568 mitotic cells were counted in each group). The cells were stained for γ‐tubulin (green), centrin2 (red), and DNA (blue). The insets show magnification of the centrin2 signal at the indicated poles. Scale bars, 5 μm. The percentage of spindle poles with n centrioles found in cells with multiple γ‐tubulin foci was assessed (411–805 poles were counted in each group). Data information: In (C and E), values (means ± s.d.) are from three independent experiments. ** P
    Figure Legend Snippet: Supernumerary centrosomes induced by ADD1 depletion result from centriole splitting during mitosis HeLa cells were infected with lentiviruses expressing shRNAs specific to ADD1 (sh‐ADD1), Aurora‐B (sh‐AurB), or luciferase (sh‐Luc.). FLAG‐ADD1 WT or mutants (S726A and S726D) were re‐expressed into the cells whose endogenous ADD1 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. The cells were stained for γ‐tubulin (red), α‐tubulin (green), and DNA (blue). Scale bars, 5 μm. The percentage of multiple γ‐tubulin foci in the total counted mitotic cells was measured (302–568 mitotic cells were counted in each group). The cells were stained for γ‐tubulin (green), centrin2 (red), and DNA (blue). The insets show magnification of the centrin2 signal at the indicated poles. Scale bars, 5 μm. The percentage of spindle poles with n centrioles found in cells with multiple γ‐tubulin foci was assessed (411–805 poles were counted in each group). Data information: In (C and E), values (means ± s.d.) are from three independent experiments. ** P

    Techniques Used: Infection, Expressing, Luciferase, Staining

    FLAG ‐ ADD 1 S726A restores the defects of spindle distortion and elongation, but not multipolar spindle formation, induced by ADD 1 depletion HeLa cells were infected with lentiviruses expressing shRNAs to ADD1 (sh‐ADD1) or luciferase (sh‐Luc.) as a control. FLAG‐ADD1 WT or the S726A mutant was re‐expressed in the cells whose endogenous ADD1 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. The percentage of distorted spindles in the total number of mitotic cells was measured (367–408 mitotic cells were counted in each group). The ratio of spindle length to cell diameter was measured (80–112 mitotic cells were counted in each group). The percentage of multipolar spindles in the total number of mitotic cells was measured (405–509 mitotic cells were counted in each group). Data information: In (B–D), values (mean ± s.d.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t ‐test: for (B), *** P = 0.0009; for (C), ** P = 0.0092; for (D), ** P = 0.0018, *** P = 0.0002. Source data are available online for this figure.
    Figure Legend Snippet: FLAG ‐ ADD 1 S726A restores the defects of spindle distortion and elongation, but not multipolar spindle formation, induced by ADD 1 depletion HeLa cells were infected with lentiviruses expressing shRNAs to ADD1 (sh‐ADD1) or luciferase (sh‐Luc.) as a control. FLAG‐ADD1 WT or the S726A mutant was re‐expressed in the cells whose endogenous ADD1 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. The percentage of distorted spindles in the total number of mitotic cells was measured (367–408 mitotic cells were counted in each group). The ratio of spindle length to cell diameter was measured (80–112 mitotic cells were counted in each group). The percentage of multipolar spindles in the total number of mitotic cells was measured (405–509 mitotic cells were counted in each group). Data information: In (B–D), values (mean ± s.d.) are from three independent experiments. Statistical significance of differences is assessed with a Student's t ‐test: for (B), *** P = 0.0009; for (C), ** P = 0.0092; for (D), ** P = 0.0018, *** P = 0.0002. Source data are available online for this figure.

    Techniques Used: Infection, Expressing, Luciferase, Mutagenesis

    ADD1 phosphorylation at S726 is important for its interaction with TPX2 HeLa cells expressing FLAG‐ADD1 WT or the S726A mutant remained asynchronized (Async.) or were synchronized at the M phase. Whole‐cell lysates were incubated with anti‐FLAG M2 affinity resins. The bound proteins were eluted from the resins with FLAG peptides and analyzed by immunoblotting (IB) with anti‐FLAG and anti‐TPX2 antibodies. WCL, whole‐cell lysates. Centrosomes were isolated from mitotic‐arrested HeLa cells using discontinuous gradient ultracentrifugation. The fractions enriched with γ‐tubulin were analyzed by immunoblotting with the indicated antibodies. HeLa cells were either placed at 4°C for 30 min (cold shock) or left at 37°C before fixation and then stained for TPX2 (green), and ADD1 pS726 (red). The arrow indicates the spindle pole region. Scale bars, 5 μm. RPE1 cells were placed at 4°C for 30 min before fixation and then stained for centrin1, TPX2, γ‐tubulin, and DNA. Scale bars, 10 μm (main image) and 1 μm (zoomed images). For the in vitro GST pull‐down assay, immobilized GST‐TPX2 fusion proteins were incubated with the cell lysates from HEK293 cells expressing FLAG‐ADD1. The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. The GST fusion proteins were visualized by Coomassie blue stain or Ponceau S stain. FLAG‐ADD1 was transiently expressed in HEK293 cells, affinity‐purified by FLAG beads, and eluted with a FLAG peptide. Immobilized GST‐TPX2 aa 120–370 fusion protein or GST alone (control) was incubated with purified FLAG‐ADD1. The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. Immobilized GST‐TPX2 aa 120–370 fusion protein or GST alone (control) was incubated with the cell lysates from HEK293 cells transiently expressing FLAG‐ADD1, the tail domain, or the mutant with a deletion at the tail domain (Δtail). The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. Immobilized GST‐TPX2 aa 120–370 fusion protein was incubated with the cell lysates from HEK293 cells transiently expressing FLAG‐ADD1 WT or the S726A mutant. The bound proteins were analyzed by immunoblotting with anti‐FLAG. Data information: Values in (A and H) are means ± s.d. Data are from three independent experiments (A) or five independent experiments (H) and expressed as the percentage relative to the level of FLAG‐ADD1 WT. ** P = 0.0091 and *** P = 0.00006 (Student's t ‐test). Source data are available online for this figure.
    Figure Legend Snippet: ADD1 phosphorylation at S726 is important for its interaction with TPX2 HeLa cells expressing FLAG‐ADD1 WT or the S726A mutant remained asynchronized (Async.) or were synchronized at the M phase. Whole‐cell lysates were incubated with anti‐FLAG M2 affinity resins. The bound proteins were eluted from the resins with FLAG peptides and analyzed by immunoblotting (IB) with anti‐FLAG and anti‐TPX2 antibodies. WCL, whole‐cell lysates. Centrosomes were isolated from mitotic‐arrested HeLa cells using discontinuous gradient ultracentrifugation. The fractions enriched with γ‐tubulin were analyzed by immunoblotting with the indicated antibodies. HeLa cells were either placed at 4°C for 30 min (cold shock) or left at 37°C before fixation and then stained for TPX2 (green), and ADD1 pS726 (red). The arrow indicates the spindle pole region. Scale bars, 5 μm. RPE1 cells were placed at 4°C for 30 min before fixation and then stained for centrin1, TPX2, γ‐tubulin, and DNA. Scale bars, 10 μm (main image) and 1 μm (zoomed images). For the in vitro GST pull‐down assay, immobilized GST‐TPX2 fusion proteins were incubated with the cell lysates from HEK293 cells expressing FLAG‐ADD1. The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. The GST fusion proteins were visualized by Coomassie blue stain or Ponceau S stain. FLAG‐ADD1 was transiently expressed in HEK293 cells, affinity‐purified by FLAG beads, and eluted with a FLAG peptide. Immobilized GST‐TPX2 aa 120–370 fusion protein or GST alone (control) was incubated with purified FLAG‐ADD1. The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. Immobilized GST‐TPX2 aa 120–370 fusion protein or GST alone (control) was incubated with the cell lysates from HEK293 cells transiently expressing FLAG‐ADD1, the tail domain, or the mutant with a deletion at the tail domain (Δtail). The bound proteins were analyzed by immunoblotting (IB) with anti‐FLAG antibody. Immobilized GST‐TPX2 aa 120–370 fusion protein was incubated with the cell lysates from HEK293 cells transiently expressing FLAG‐ADD1 WT or the S726A mutant. The bound proteins were analyzed by immunoblotting with anti‐FLAG. Data information: Values in (A and H) are means ± s.d. Data are from three independent experiments (A) or five independent experiments (H) and expressed as the percentage relative to the level of FLAG‐ADD1 WT. ** P = 0.0091 and *** P = 0.00006 (Student's t ‐test). Source data are available online for this figure.

    Techniques Used: Expressing, Mutagenesis, Incubation, Isolation, Staining, In Vitro, Pull Down Assay, Affinity Purification, Purification

    ADD1–TPX2 interaction is important for bipolar spindle formation HA‐tagged TPX2 WT (HA‐TPX2 WT) and the deletion mutants (HA‐TPX2 Δ1–43, ∆236–370 and ∆711–747) were transiently expressed in HEK293 cells and then analyzed by immunoblotting (IB) with anti‐TPX2 and anti‐HA. HeLa cells were infected with lentiviruses expressing shRNAs to TPX2 (sh‐TPX2), or luciferase (sh‐Luc.). HA‐TPX2 WT or the mutants (∆1–43, ∆236–370 and ∆711–747) were re‐expressed in the cells whose endogenous TPX2 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. FLAG‐tagged ADD1 (FLAG‐ADD1) or Aurora‐A (FLAG‐AurA) was transiently co‐expressed with HA‐TPX2 or its mutants in HEK293 cells. FLAG‐tagged proteins were immunoprecipitated (IP) by anti‐FLAG, and the immunocomplexes were analyzed by immunoblotting with anti‐FLAG or anti‐HA antibody. The cells were stained for TPX2 (green), α‐tubulin (red), and DNA (blue). Scale bars, 5 μm. The ratio of spindle length to cell diameter was measured (116–244 mitotic cells were counted in each group). The percentage of multipolar spindles in the total counted mitotic cells was measured (299–1,323 mitotic cells were counted in each group). Data information: Values (means ± s.d.) are from three independent experiments. * P
    Figure Legend Snippet: ADD1–TPX2 interaction is important for bipolar spindle formation HA‐tagged TPX2 WT (HA‐TPX2 WT) and the deletion mutants (HA‐TPX2 Δ1–43, ∆236–370 and ∆711–747) were transiently expressed in HEK293 cells and then analyzed by immunoblotting (IB) with anti‐TPX2 and anti‐HA. HeLa cells were infected with lentiviruses expressing shRNAs to TPX2 (sh‐TPX2), or luciferase (sh‐Luc.). HA‐TPX2 WT or the mutants (∆1–43, ∆236–370 and ∆711–747) were re‐expressed in the cells whose endogenous TPX2 had been depleted. Equal amounts of whole‐cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. FLAG‐tagged ADD1 (FLAG‐ADD1) or Aurora‐A (FLAG‐AurA) was transiently co‐expressed with HA‐TPX2 or its mutants in HEK293 cells. FLAG‐tagged proteins were immunoprecipitated (IP) by anti‐FLAG, and the immunocomplexes were analyzed by immunoblotting with anti‐FLAG or anti‐HA antibody. The cells were stained for TPX2 (green), α‐tubulin (red), and DNA (blue). Scale bars, 5 μm. The ratio of spindle length to cell diameter was measured (116–244 mitotic cells were counted in each group). The percentage of multipolar spindles in the total counted mitotic cells was measured (299–1,323 mitotic cells were counted in each group). Data information: Values (means ± s.d.) are from three independent experiments. * P

    Techniques Used: Infection, Expressing, Luciferase, Immunoprecipitation, Staining

    26) Product Images from "The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation *"

    Article Title: The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.497297

    Catalytically inactive r-VKORC1(C132A/C135A) FLAG shows a dominant negative effect on KO reduction in cells and carboxylation in vitro . 293 cells expressing only endogenous VKORC1 or also expressing r-VKORC1(C132A/C135A) FLAG were assayed for KO reduction
    Figure Legend Snippet: Catalytically inactive r-VKORC1(C132A/C135A) FLAG shows a dominant negative effect on KO reduction in cells and carboxylation in vitro . 293 cells expressing only endogenous VKORC1 or also expressing r-VKORC1(C132A/C135A) FLAG were assayed for KO reduction

    Techniques Used: Dominant Negative Mutation, In Vitro, Expressing

    Purified VKORC1 efficiently reduces KO to KH 2 . Samples purified from insect cells infected with baculovirus containing VKORC1 FLAG ( a ) or from mock-infected cells ( b ) were incubated with KO, followed by vitamin K isolation, all under nitrogen in sealed
    Figure Legend Snippet: Purified VKORC1 efficiently reduces KO to KH 2 . Samples purified from insect cells infected with baculovirus containing VKORC1 FLAG ( a ) or from mock-infected cells ( b ) were incubated with KO, followed by vitamin K isolation, all under nitrogen in sealed

    Techniques Used: Purification, Infection, Incubation, Isolation

    VKORC1 reduces vitamin K epoxide to hydroquinone to drive carboxylation. Microsomes were prepared from insect cells coinfected with baculoviruses containing r-VKORC1 FLAG (VKOR) and r-carboxylase ( Carb ) or infected only with baculovirus containing the
    Figure Legend Snippet: VKORC1 reduces vitamin K epoxide to hydroquinone to drive carboxylation. Microsomes were prepared from insect cells coinfected with baculoviruses containing r-VKORC1 FLAG (VKOR) and r-carboxylase ( Carb ) or infected only with baculovirus containing the

    Techniques Used: Infection

    An assay to monitor KO reduction in cells. 293 cells ( a ) and r-wild type (wt) VKORC1 FLAG 293 cells ( b ) were incubated with KO, and vitamin K was subsequently extracted and analyzed by HPLC to monitor KO reduction to K. Similar results were obtained in
    Figure Legend Snippet: An assay to monitor KO reduction in cells. 293 cells ( a ) and r-wild type (wt) VKORC1 FLAG 293 cells ( b ) were incubated with KO, and vitamin K was subsequently extracted and analyzed by HPLC to monitor KO reduction to K. Similar results were obtained in

    Techniques Used: Incubation, High Performance Liquid Chromatography

    Endogenous VKORC1 up-regulation in cells expressing a r- FLAG VKORC1. Equivalent amounts of lysate (25 μg) prepared from cells expressing only endogenous VKORC1 or also expressing N-terminally FLAG-tagged r-VKORC1 were assayed by Western analysis
    Figure Legend Snippet: Endogenous VKORC1 up-regulation in cells expressing a r- FLAG VKORC1. Equivalent amounts of lysate (25 μg) prepared from cells expressing only endogenous VKORC1 or also expressing N-terminally FLAG-tagged r-VKORC1 were assayed by Western analysis

    Techniques Used: Expressing, Western Blot

    Catalytically inactive r-VKORC1(C132A/C135A) FLAG associates with wild type endogenous VKORC1 in 293 cells. a , r-VKORC1(C132A/C135A) FLAG was overexpressed 5- or 7-fold in 293 cells (clones 1 and 2, respectively). b–d , the mutant in clone 1 was
    Figure Legend Snippet: Catalytically inactive r-VKORC1(C132A/C135A) FLAG associates with wild type endogenous VKORC1 in 293 cells. a , r-VKORC1(C132A/C135A) FLAG was overexpressed 5- or 7-fold in 293 cells (clones 1 and 2, respectively). b–d , the mutant in clone 1 was

    Techniques Used: Mutagenesis

    Expression of r-wild type VKORC1 FLAG in 293 cells reveals association with endogenous VKORC1. Equivalent amounts of lysates (25 μg) from 293 cells expressing endogenous VKORC1 or also expressing r-wild type VKORC1 FLAG were assayed by Western analysis
    Figure Legend Snippet: Expression of r-wild type VKORC1 FLAG in 293 cells reveals association with endogenous VKORC1. Equivalent amounts of lysates (25 μg) from 293 cells expressing endogenous VKORC1 or also expressing r-wild type VKORC1 FLAG were assayed by Western analysis

    Techniques Used: Expressing, Western Blot

    Purification of r-VKORC1 FLAG from insect cells. a , fractions were monitored in a Western blot using anti-VKORC1 antibody. b , purified VKORC1 FLAG was also analyzed by SDS-PAGE and Coomassie staining. VKORC1 ( V , empty arrowheads ), and a small amount of
    Figure Legend Snippet: Purification of r-VKORC1 FLAG from insect cells. a , fractions were monitored in a Western blot using anti-VKORC1 antibody. b , purified VKORC1 FLAG was also analyzed by SDS-PAGE and Coomassie staining. VKORC1 ( V , empty arrowheads ), and a small amount of

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

    27) Product Images from "AAA Peroxins and Their Recruiter Pex26p Modulate the Interactions of Peroxins Involved in Peroxisomal Protein Import *"

    Article Title: AAA Peroxins and Their Recruiter Pex26p Modulate the Interactions of Peroxins Involved in Peroxisomal Protein Import *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.588038

    AAA cassettes of Pex1p and Pex6p are involved in the dissociation of the Pex26p·Pex14p complex. A , FLAG-Pex26p was expressed in pex1 ZP107 together with Pex1p-HA and Pex6p-HA ( lane 1 ) as well as Pex14p ( lane 2 ) as indicated at the top . AAA cassette
    Figure Legend Snippet: AAA cassettes of Pex1p and Pex6p are involved in the dissociation of the Pex26p·Pex14p complex. A , FLAG-Pex26p was expressed in pex1 ZP107 together with Pex1p-HA and Pex6p-HA ( lane 1 ) as well as Pex14p ( lane 2 ) as indicated at the top . AAA cassette

    Techniques Used:

    Direct binding of Pex1p to Pex5p. Aa , SDS-PAGE analysis of purified FLAG-Pex1p ( lane 1 ), His-Pex5p ( lane 2 ), and His-Pex14p ( lane 3 ). b , homo-oligomer of recombinant FLAG-Pex1p. FLAG-Pex1p was verified for oligomerization by BN-PAGE. Staining was done
    Figure Legend Snippet: Direct binding of Pex1p to Pex5p. Aa , SDS-PAGE analysis of purified FLAG-Pex1p ( lane 1 ), His-Pex5p ( lane 2 ), and His-Pex14p ( lane 3 ). b , homo-oligomer of recombinant FLAG-Pex1p. FLAG-Pex1p was verified for oligomerization by BN-PAGE. Staining was done

    Techniques Used: Binding Assay, SDS Page, Purification, Recombinant, Polyacrylamide Gel Electrophoresis, Staining

    Pex1p·Pex6p complex dissociates Pex26p bound to Pex14p. Wild type or mutants of FLAG-Pex26p were expressed in pex26 ZP167 together (+) with Pex1p-HA and Pex6p-HA ( lanes 1 , 4 , 7 , 10 , and 13 ), Pex14p ( lanes 2 , 5 , 8 , 11 , and 14 ), or Pex1p-HA and
    Figure Legend Snippet: Pex1p·Pex6p complex dissociates Pex26p bound to Pex14p. Wild type or mutants of FLAG-Pex26p were expressed in pex26 ZP167 together (+) with Pex1p-HA and Pex6p-HA ( lanes 1 , 4 , 7 , 10 , and 13 ), Pex14p ( lanes 2 , 5 , 8 , 11 , and 14 ), or Pex1p-HA and

    Techniques Used:

    28) Product Images from "A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1"

    Article Title: A quantitative RNA code for mRNA target selection by the germline fate determinant GLD-1

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2010.334

    Identification of mRNAs associated with GLD-1. ( A ) GLD-1 is expressed in the medial gonad and is a key regulator of germline development, yet the majority of its biological targets are unknown. ‘Distal-most', ‘medial', and ‘proximal' indicate parts of a wild-type adult worm gonad. The asterisk here and in subsequent figures indicates the distal end of the gonad. ( B ) Experimental outline of GLD-1 mRNA target identification. Two independent strategies were employed: (1) comparison of anti-FLAG IP (aFLAG) to anti-MYC IP (a MYC) on extract from worms expressing tagged GLD-1 (left panel); (2) comparison of anti-FLAG IP on extract from worms expressing tagged GLD-1 (GGF_IP) to anti-FLAG IP on extract from non-tagged (N2) worms (N2_IP) (right panel). To ensure that transcripts were detected with high confidence, an input aliquot (RNA purified from extract before IP) from strategy 1 was initially used to establish an input cutoff ( > 5.5). ( C ) A large set of mRNAs (red dots) are enriched greater than three-fold in a GLD 1 IP. Comparison of aFLAG to the corresponding input. ( D ) GLD-1-associated mRNAs (red dots, as in C ) are not enriched in a control IP. Comparison of aMYC to the corresponding input. ( E ) The enrichment of associated mRNAs is reproducible between complementary GLD-1 IP approaches. Comparison of aFLAG—aMYC ( C , D ) on the x axis, to GGF_IP—N2_IP on the y .
    Figure Legend Snippet: Identification of mRNAs associated with GLD-1. ( A ) GLD-1 is expressed in the medial gonad and is a key regulator of germline development, yet the majority of its biological targets are unknown. ‘Distal-most', ‘medial', and ‘proximal' indicate parts of a wild-type adult worm gonad. The asterisk here and in subsequent figures indicates the distal end of the gonad. ( B ) Experimental outline of GLD-1 mRNA target identification. Two independent strategies were employed: (1) comparison of anti-FLAG IP (aFLAG) to anti-MYC IP (a MYC) on extract from worms expressing tagged GLD-1 (left panel); (2) comparison of anti-FLAG IP on extract from worms expressing tagged GLD-1 (GGF_IP) to anti-FLAG IP on extract from non-tagged (N2) worms (N2_IP) (right panel). To ensure that transcripts were detected with high confidence, an input aliquot (RNA purified from extract before IP) from strategy 1 was initially used to establish an input cutoff ( > 5.5). ( C ) A large set of mRNAs (red dots) are enriched greater than three-fold in a GLD 1 IP. Comparison of aFLAG to the corresponding input. ( D ) GLD-1-associated mRNAs (red dots, as in C ) are not enriched in a control IP. Comparison of aMYC to the corresponding input. ( E ) The enrichment of associated mRNAs is reproducible between complementary GLD-1 IP approaches. Comparison of aFLAG—aMYC ( C , D ) on the x axis, to GGF_IP—N2_IP on the y .

    Techniques Used: Expressing, Purification

    29) Product Images from "USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock"

    Article Title: USP7 and TDP-43: Pleiotropic Regulation of Cryptochrome Protein Stability Paces the Oscillation of the Mammalian Circadian Clock

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154263

    USP7 interacts with CRY proteins. A. Silver staining image of proteins co-purified with FLAG-His-Myc-CRY1 (FHM-CRY1) or FHM-CRY2. NIH3T3 cells expressing FHM-CRY1 or FHM-CRY2 were treated with 10 μM MG132 for 6 hours and lysed with IP Buffer. Cell lysates were subjected to immunoprecipitation using anti-FLAG-M2 agarose beads. FH-LacZ expressed in NIH3T3 cells was used as a control. B . The numbers of proteins co-purified with FHM-CRY1 or FHM-CRY2. Proteins co-purified with FH-LacZ were eliminated from the list of CRY1 and CRY2 interacting proteins. Proteins detected in both CRY1 and CRY2 samples with high MS scores were listed in S1 Table . C. Interaction of USP7 with CRY2 protein. NIH3T3 cells expressing FLAG-CRY2 and/or Myc-USP7 were cultured in the presence of 10 μM MG132 for 6 hours and lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-FLAG, anti-Myc antibody or normal mouse IgG (negative control) as precipitating antibodies.
    Figure Legend Snippet: USP7 interacts with CRY proteins. A. Silver staining image of proteins co-purified with FLAG-His-Myc-CRY1 (FHM-CRY1) or FHM-CRY2. NIH3T3 cells expressing FHM-CRY1 or FHM-CRY2 were treated with 10 μM MG132 for 6 hours and lysed with IP Buffer. Cell lysates were subjected to immunoprecipitation using anti-FLAG-M2 agarose beads. FH-LacZ expressed in NIH3T3 cells was used as a control. B . The numbers of proteins co-purified with FHM-CRY1 or FHM-CRY2. Proteins co-purified with FH-LacZ were eliminated from the list of CRY1 and CRY2 interacting proteins. Proteins detected in both CRY1 and CRY2 samples with high MS scores were listed in S1 Table . C. Interaction of USP7 with CRY2 protein. NIH3T3 cells expressing FLAG-CRY2 and/or Myc-USP7 were cultured in the presence of 10 μM MG132 for 6 hours and lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-FLAG, anti-Myc antibody or normal mouse IgG (negative control) as precipitating antibodies.

    Techniques Used: Silver Staining, Purification, Expressing, Immunoprecipitation, Mass Spectrometry, Cell Culture, Negative Control

    USP7 deubiquitinates CRY proteins. A . In vitro ubiquitination assay. HEK293T/17 cells were transfected with the expression vector of FLAG-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then harvested. FLAG-CRY2 purified from the cell lysate with anti-FLAG M2 agarose beads was incubated with or without a recombinant protein, full-length USP7 or a catalytic domain of USP2 (USP2 CD), for 30 min at 37°C. Recombinant USP2 catalytic domain was used as a positive control [ 26 ]. B . In vivo deubiquitination assay in HEK293T/17 cells. The cells were transfected with indicated expression vectors. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then lysed with IP Buffer. FLAG-CRY2 was purified with anti-FLAG M2 agarose beads, followed by western blotting analysis with anti-CRY2 antibody. An inactive mutant of USP7 (USP7-C223A) was used for a negative control. C. Effect of USP7-specific inhibitor on CRY up-shifted bands. NIH3T3 cells were transfected with the expression vector for Myc-CRY1 or Myc-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 20 μM HBX 41108 for 6 hours. The smear bands of Myc-CRY1 or Myc-CRY2 were quantified (means + SEM, n = 3, **: p
    Figure Legend Snippet: USP7 deubiquitinates CRY proteins. A . In vitro ubiquitination assay. HEK293T/17 cells were transfected with the expression vector of FLAG-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then harvested. FLAG-CRY2 purified from the cell lysate with anti-FLAG M2 agarose beads was incubated with or without a recombinant protein, full-length USP7 or a catalytic domain of USP2 (USP2 CD), for 30 min at 37°C. Recombinant USP2 catalytic domain was used as a positive control [ 26 ]. B . In vivo deubiquitination assay in HEK293T/17 cells. The cells were transfected with indicated expression vectors. Forty-two hours after the transfection, the cells were cultured in the presence of 10 μM MG132 for 6 hours and then lysed with IP Buffer. FLAG-CRY2 was purified with anti-FLAG M2 agarose beads, followed by western blotting analysis with anti-CRY2 antibody. An inactive mutant of USP7 (USP7-C223A) was used for a negative control. C. Effect of USP7-specific inhibitor on CRY up-shifted bands. NIH3T3 cells were transfected with the expression vector for Myc-CRY1 or Myc-CRY2. Forty-two hours after the transfection, the cells were cultured in the presence of 20 μM HBX 41108 for 6 hours. The smear bands of Myc-CRY1 or Myc-CRY2 were quantified (means + SEM, n = 3, **: p

    Techniques Used: In Vitro, Ubiquitin Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Purification, Incubation, Recombinant, Positive Control, In Vivo, Western Blot, Mutagenesis, Negative Control

    TDP-43 interacts with CRY proteins and stabilizes CRY proteins. A. Effect of TDP-43 expression on CRY protein levels. HEK293T/17 cells were transfected with expression vectors for Myc-CRY1, Myc-CRY2 and FLAG-TDP-43, and cultured for 48 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. Quantified data are shown by means + SEM (n = 3, **: p
    Figure Legend Snippet: TDP-43 interacts with CRY proteins and stabilizes CRY proteins. A. Effect of TDP-43 expression on CRY protein levels. HEK293T/17 cells were transfected with expression vectors for Myc-CRY1, Myc-CRY2 and FLAG-TDP-43, and cultured for 48 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. Quantified data are shown by means + SEM (n = 3, **: p

    Techniques Used: Expressing, Transfection, Cell Culture, SDS Page, Western Blot

    30) Product Images from "Prestin Surface Expression and Activity Are Augmented by Interaction with MAP1S, a Microtubule-associated Protein *"

    Article Title: Prestin Surface Expression and Activity Are Augmented by Interaction with MAP1S, a Microtubule-associated Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.117853

    Reciprocal immunoprecipitations confirm the interaction between prestin and MAP1S. A permanent cell line expressing prestin-YFP-myc was transfected with FLAG-MAP1S-CFP. A , immunoprecipitations were performed with anti-FLAG (MAP1S) antibody and the presence
    Figure Legend Snippet: Reciprocal immunoprecipitations confirm the interaction between prestin and MAP1S. A permanent cell line expressing prestin-YFP-myc was transfected with FLAG-MAP1S-CFP. A , immunoprecipitations were performed with anti-FLAG (MAP1S) antibody and the presence

    Techniques Used: Expressing, Transfection

    31) Product Images from "Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins"

    Article Title: Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

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

    doi: 10.1073/pnas.1304231110

    NEIL1 is more efficient in LP-BER than NEIL2 when normalized to the same specific glycosylase activity. ( A ) FLAG IPs of FLAG-tagged NEIL1 and NEIL2 were adjusted for equal 5-OHU excision activity, based on the amount of protein by Western blotting with
    Figure Legend Snippet: NEIL1 is more efficient in LP-BER than NEIL2 when normalized to the same specific glycosylase activity. ( A ) FLAG IPs of FLAG-tagged NEIL1 and NEIL2 were adjusted for equal 5-OHU excision activity, based on the amount of protein by Western blotting with

    Techniques Used: Activity Assay, Western Blot

    32) Product Images from "Molecular Basis of Zinc-Dependent Endocytosis of Human ZIP4 Transceptor"

    Article Title: Molecular Basis of Zinc-Dependent Endocytosis of Human ZIP4 Transceptor

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.107582

    Partial Proteolysis of hZIP4 (A) The FLAG-hZIP4-HA construct. The fragment in red is the peptide identified in mass spectrometry ( Figure S5 ). The LQL motif is in bold. SP, signal peptide of hZIP4 (residues 1–22). (B) Partial proteolysis of purified hZIP4 by chymotrypsin under optimized condition. Proteolysis products were detected by Coomassie blue staining and western blots using antibodies against FLAG tag or HA tag. (C) Effects of zinc ions on proteolysis of the wild-type hZIP4 (left) and the D511A mutant (right) detected by western blot against the HA tag. The quantitative analysis of zinc protection for the wild-type protein is shown in Figure S6 .
    Figure Legend Snippet: Partial Proteolysis of hZIP4 (A) The FLAG-hZIP4-HA construct. The fragment in red is the peptide identified in mass spectrometry ( Figure S5 ). The LQL motif is in bold. SP, signal peptide of hZIP4 (residues 1–22). (B) Partial proteolysis of purified hZIP4 by chymotrypsin under optimized condition. Proteolysis products were detected by Coomassie blue staining and western blots using antibodies against FLAG tag or HA tag. (C) Effects of zinc ions on proteolysis of the wild-type hZIP4 (left) and the D511A mutant (right) detected by western blot against the HA tag. The quantitative analysis of zinc protection for the wild-type protein is shown in Figure S6 .

    Techniques Used: Construct, Mass Spectrometry, Purification, Staining, Western Blot, FLAG-tag, Mutagenesis

    33) Product Images from "Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex"

    Article Title: Pax7 activates myogenic genes by recruitment of a histone methyltransferase complex

    Journal: Nature cell biology

    doi: 10.1038/ncb1671

    Identification of Pax7-interacting co-factors. ( a ) A TAP tag, consisting of six histidine and three FLAG epitopes, separated by a TEV cleavage site, was fused to the C-terminus of Pax7 to create a Pax7–CTAP (His–TEV–FLAG) construct. A construct expressing only the tag (referred to as HisFLAG-tag) was used as a negative control. ( b ) High yield purification of the Pax7–CTAP protein as shown by detection of the fusion protein in the initial cell lysate, in the eluate following TEV cleavage, and in the final elution from Ni + -resin. ( c, ) TAP of Pax7–CTAP- compared with HisFLAG-tag-associated proteins from C2C12 cells. Protein matches were generated from multiple data sets and were identified following MALDI–TOF (analysed via Mascot). Pax7–CTAP-interacting proteins were compared with those identified in HisFLAG-tag purifications to unequivocally identify those that were Pax7-specific (versus those that represented contaminants). ( d ) Pax7 co-immunoprecipitated with Wdr5 and Ash2L, two conserved units of a HMT complex. As a negative control, the Pax7-immunoprecipitate was also probed with an antibody to a non-native-Pax7 interacting nuclear protein, ERK1/2. ( e ) Co-purification of Pax7 and MLL2 immunoprecipitated from primary myoblast nuclear extract. ( f ) Pax7-deletion constructs used to map Pax7 binding to the HMT complex. ( g ) Loss of the paired domain was observed to almost entirely abolish binding between Pax7 and Wdr5. ( h .
    Figure Legend Snippet: Identification of Pax7-interacting co-factors. ( a ) A TAP tag, consisting of six histidine and three FLAG epitopes, separated by a TEV cleavage site, was fused to the C-terminus of Pax7 to create a Pax7–CTAP (His–TEV–FLAG) construct. A construct expressing only the tag (referred to as HisFLAG-tag) was used as a negative control. ( b ) High yield purification of the Pax7–CTAP protein as shown by detection of the fusion protein in the initial cell lysate, in the eluate following TEV cleavage, and in the final elution from Ni + -resin. ( c, ) TAP of Pax7–CTAP- compared with HisFLAG-tag-associated proteins from C2C12 cells. Protein matches were generated from multiple data sets and were identified following MALDI–TOF (analysed via Mascot). Pax7–CTAP-interacting proteins were compared with those identified in HisFLAG-tag purifications to unequivocally identify those that were Pax7-specific (versus those that represented contaminants). ( d ) Pax7 co-immunoprecipitated with Wdr5 and Ash2L, two conserved units of a HMT complex. As a negative control, the Pax7-immunoprecipitate was also probed with an antibody to a non-native-Pax7 interacting nuclear protein, ERK1/2. ( e ) Co-purification of Pax7 and MLL2 immunoprecipitated from primary myoblast nuclear extract. ( f ) Pax7-deletion constructs used to map Pax7 binding to the HMT complex. ( g ) Loss of the paired domain was observed to almost entirely abolish binding between Pax7 and Wdr5. ( h .

    Techniques Used: Construct, Expressing, Negative Control, Purification, Generated, Immunoprecipitation, HMT Assay, Copurification, Binding Assay

    34) Product Images from "Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C"

    Article Title: Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.110.070573

    NPR-C is rapidly and constitutively internalized in HeLa and 293 cells. A, tTA-HeLa cells were transiently transfected with FLAG-NPR-C. Cells were then labeled with anti-FLAG antibody followed by anti-mouse 125 I-IgG at 4°C. Cells were incubated at 37°C for the indicated times before acid washing. Values represent the average ± S.E.M., where n = 14. B, 293 cells were transfected with FLAG-NPR-C and labeled with either 125 I-ANP or 125 I-IgG at 4°C. Aliquots were incubated at 37°C for the times shown and before acid washing. Values represent the average ± S.E.M., where n = 6. C, 293 cells transiently transfected with FLAG-NPR-C were labeled with anti-FLAG antibody and 125 I-IgG secondary antibody at 4°C. Aliquots were incubated at 37°C in the presence or absence of 1 μM ANP, BNP, or CNP for the times indicated, where n = 4.
    Figure Legend Snippet: NPR-C is rapidly and constitutively internalized in HeLa and 293 cells. A, tTA-HeLa cells were transiently transfected with FLAG-NPR-C. Cells were then labeled with anti-FLAG antibody followed by anti-mouse 125 I-IgG at 4°C. Cells were incubated at 37°C for the indicated times before acid washing. Values represent the average ± S.E.M., where n = 14. B, 293 cells were transfected with FLAG-NPR-C and labeled with either 125 I-ANP or 125 I-IgG at 4°C. Aliquots were incubated at 37°C for the times shown and before acid washing. Values represent the average ± S.E.M., where n = 6. C, 293 cells transiently transfected with FLAG-NPR-C were labeled with anti-FLAG antibody and 125 I-IgG secondary antibody at 4°C. Aliquots were incubated at 37°C in the presence or absence of 1 μM ANP, BNP, or CNP for the times indicated, where n = 4.

    Techniques Used: Transfection, Labeling, Incubation, Aqueous Normal-phase Chromatography

    GC-A is rapidly internalized in 293 PMA cells. A, 293 cells stably expressing FLAG-GC-A were incubated at 4°C with either 125 I-ANP or anti-FLAG antibody followed by anti-mouse 125 I-IgG. Cells were incubated at 37°C for the indicated times before acid-washing and counting. Values represent average ± S.E.M., where n = 14. B, untransfected 293 cells or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with 125 I-ANP. Internalized radioactivity as a function of time at 37°C is shown. Values represent average ± the range of two determinations. C, untransfected or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with anti-FLAG antibody followed by 125 I-IgG secondary antibody. The cells were incubated at 37°C in the presence or absence of 1 μM ANP for the indicated periods of time. Values represent the average ± the range determinations, where n = 2.
    Figure Legend Snippet: GC-A is rapidly internalized in 293 PMA cells. A, 293 cells stably expressing FLAG-GC-A were incubated at 4°C with either 125 I-ANP or anti-FLAG antibody followed by anti-mouse 125 I-IgG. Cells were incubated at 37°C for the indicated times before acid-washing and counting. Values represent average ± S.E.M., where n = 14. B, untransfected 293 cells or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with 125 I-ANP. Internalized radioactivity as a function of time at 37°C is shown. Values represent average ± the range of two determinations. C, untransfected or 293 cells stably expressing FLAG-GC-A were labeled at 4°C with anti-FLAG antibody followed by 125 I-IgG secondary antibody. The cells were incubated at 37°C in the presence or absence of 1 μM ANP for the indicated periods of time. Values represent the average ± the range determinations, where n = 2.

    Techniques Used: Stable Transfection, Expressing, Incubation, Aqueous Normal-phase Chromatography, Labeling, Radioactivity

    A, 125 I-ANP uptake in tTA-HeLa cells transiently transfected with GFP or FLAG-GC-A. Cells were labeled with subsaturating concentrations of 125 I-ANP at 4°C. Aliquots of labeled cells were incubated at 37°C for the times indicated before acid washing and counting. Values represent average ± the range of the determinations, where n = 2. The graph is representative of multiple experiments. B, FLAG-GC-A and wild-type GC-A bind and are activated by ANP similarly. The 293 cells were transiently transfected with wild-type GC-A or FLAG-GC-A and incubated with increasing concentrations of ANP for 1 min. Cellular cGMP concentrations were measured and plotted as a function of peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate. C, FLAG-GC-A has similar affinity for ANP as wild-type GC-A. Transiently transfected 293 cells were incubated for 1 h at 4°C with 125 I-ANP in the presence or absence of increasing concentrations of unlabeled ligand. Specifically bound 125 I-ANP was plotted as a function of competing peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate.
    Figure Legend Snippet: A, 125 I-ANP uptake in tTA-HeLa cells transiently transfected with GFP or FLAG-GC-A. Cells were labeled with subsaturating concentrations of 125 I-ANP at 4°C. Aliquots of labeled cells were incubated at 37°C for the times indicated before acid washing and counting. Values represent average ± the range of the determinations, where n = 2. The graph is representative of multiple experiments. B, FLAG-GC-A and wild-type GC-A bind and are activated by ANP similarly. The 293 cells were transiently transfected with wild-type GC-A or FLAG-GC-A and incubated with increasing concentrations of ANP for 1 min. Cellular cGMP concentrations were measured and plotted as a function of peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate. C, FLAG-GC-A has similar affinity for ANP as wild-type GC-A. Transiently transfected 293 cells were incubated for 1 h at 4°C with 125 I-ANP in the presence or absence of increasing concentrations of unlabeled ligand. Specifically bound 125 I-ANP was plotted as a function of competing peptide concentration. The data points represent the mean ± S.E.M. assayed in triplicate.

    Techniques Used: Aqueous Normal-phase Chromatography, Transfection, Labeling, Incubation, Concentration Assay

    The 125 I-IgG uptake assay specifically measures FLAG-GC-A internalization. A, tTA-Hela cells were transiently transfected with GFP or FLAG-GCA. The cells were dispensed into tubes and incubated with 0.05 (1× primary) or 0.1 μl (20× primary) of anti-FLAG-M2 antibody. Excess antibody was removed before addition of 5 (1×), 50 (10× secondary), or 100 μl of 125 I-IgG. Cellular radioactivity was measured directly or after acid-stripping to remove surface 125 I-sIgG. Values represent the range of determinations, where n = 2. The graph is representative of more than three experiments. B, 293 cells were transiently transfected with 10 (1×), 1, or 5 μg of FLAG-GC-A plasmid DNA. Internalization assays were performed 48 h later. An equal number of cells from each transfection were separated by SDS-PAGE, blotted to an Immobilon membrane, and GC-A expression was detected by Western blot using an anti-GC-A antibody (inset). Values represent average ± S.E.M., where n = 6.
    Figure Legend Snippet: The 125 I-IgG uptake assay specifically measures FLAG-GC-A internalization. A, tTA-Hela cells were transiently transfected with GFP or FLAG-GCA. The cells were dispensed into tubes and incubated with 0.05 (1× primary) or 0.1 μl (20× primary) of anti-FLAG-M2 antibody. Excess antibody was removed before addition of 5 (1×), 50 (10× secondary), or 100 μl of 125 I-IgG. Cellular radioactivity was measured directly or after acid-stripping to remove surface 125 I-sIgG. Values represent the range of determinations, where n = 2. The graph is representative of more than three experiments. B, 293 cells were transiently transfected with 10 (1×), 1, or 5 μg of FLAG-GC-A plasmid DNA. Internalization assays were performed 48 h later. An equal number of cells from each transfection were separated by SDS-PAGE, blotted to an Immobilon membrane, and GC-A expression was detected by Western blot using an anti-GC-A antibody (inset). Values represent average ± S.E.M., where n = 6.

    Techniques Used: Transfection, Incubation, Radioactivity, Stripping Membranes, Plasmid Preparation, SDS Page, Expressing, Western Blot

    GC-A is slowly internalized in HeLa cells. A, tTA-HeLa cells transiently transfected with FLAG-GC-A were labeled with 125 I-ANP or anti-FLAG-M2 antibody followed by anti-mouse 125 I-IgG at 4°C. Values represent average ± S.E.M. where n = 4 (B) ANP increases GC-A uptake at longer but not shorter periods of time. Cells transfected with FLAG-GC-A were labeled with anti-FLAG-M2 antibody and anti-mouse 125 I-IgG before incubation at 37°C in the absence or presence of 1 μM ANP for the periods of time shown. Samples were then acid-washed and counted. Values represent average ± S.E.M., where n = 6. C, 125 I-transferrin is rapidly internalized in tTA-HeLa cells. Aliquots of the tTA-HeLa cells transfected with FLAG-GC-A were labeled with 125 I-transferrin at 4°C. Internalized 125 I-transferrin was determined after the indicated periods of time at 37°C. Values represent the average ± S.E.M., where n = 8.
    Figure Legend Snippet: GC-A is slowly internalized in HeLa cells. A, tTA-HeLa cells transiently transfected with FLAG-GC-A were labeled with 125 I-ANP or anti-FLAG-M2 antibody followed by anti-mouse 125 I-IgG at 4°C. Values represent average ± S.E.M. where n = 4 (B) ANP increases GC-A uptake at longer but not shorter periods of time. Cells transfected with FLAG-GC-A were labeled with anti-FLAG-M2 antibody and anti-mouse 125 I-IgG before incubation at 37°C in the absence or presence of 1 μM ANP for the periods of time shown. Samples were then acid-washed and counted. Values represent average ± S.E.M., where n = 6. C, 125 I-transferrin is rapidly internalized in tTA-HeLa cells. Aliquots of the tTA-HeLa cells transfected with FLAG-GC-A were labeled with 125 I-transferrin at 4°C. Internalized 125 I-transferrin was determined after the indicated periods of time at 37°C. Values represent the average ± S.E.M., where n = 8.

    Techniques Used: Transfection, Labeling, Aqueous Normal-phase Chromatography, Incubation

    Concomitant down-regulation of extracellular and intracellular FLAG-GC-A in 293 cells. The 293 cells stably expressing FLAG-GC-A were incubated with 10 μg/ml cycloheximide in the absence or presence of 200 nM ANP for the period of times indicated. In one experiment, crude membranes were prepared and then assayed for guanylyl cyclase activity in the presence of 1% Triton X-100 and Mn 2+ -GTP. In a second experiment, cells were incubated with ANP as described above and then labeled with anti-FLAG antibody followed by 125 I-IgG at 4°C. Total 125 I-IgG radioactivity and guanylyl cyclase activities were normalized to activities obtained from cells not incubated with ANP (control) and plotted as a function of time of ANP exposure.
    Figure Legend Snippet: Concomitant down-regulation of extracellular and intracellular FLAG-GC-A in 293 cells. The 293 cells stably expressing FLAG-GC-A were incubated with 10 μg/ml cycloheximide in the absence or presence of 200 nM ANP for the period of times indicated. In one experiment, crude membranes were prepared and then assayed for guanylyl cyclase activity in the presence of 1% Triton X-100 and Mn 2+ -GTP. In a second experiment, cells were incubated with ANP as described above and then labeled with anti-FLAG antibody followed by 125 I-IgG at 4°C. Total 125 I-IgG radioactivity and guanylyl cyclase activities were normalized to activities obtained from cells not incubated with ANP (control) and plotted as a function of time of ANP exposure.

    Techniques Used: Stable Transfection, Expressing, Incubation, Aqueous Normal-phase Chromatography, Activity Assay, Labeling, Radioactivity

    35) Product Images from "Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex"

    Article Title: Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex

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

    doi: 10.1073/pnas.1000681107

    Purification of Drosophila NELF. ( A ) Nuclear extracts were prepared from transgenic fly embryos expressing FLAG-NELF subunits and fractionated using anti-FLAG Sepharose (Sigma). Eluates (10 uL) were analyzed by SDS-PAGE and Coomassie staining (lanes 2–4). Arrows indicate positions of the NELF subunits. Lane 1 (Control) shows 10 uL of eluate from a mock purification of nuclear extract from nontransgenic embryos. Asterisks denote proteins that bind nonspecifically to the FLAG column. ( B ) Western blot analysis of the FLAG NELF-D complex using NELF-A, NELF-B, NELF-D, and NELF-E antibodies. ( C ) Silver-stained gel with Pol II purified from Drosophila embryo nuclear extract and Coomassie blue-stained gel with DSIF purified from baculovirus. The prominent band migrating just above Rpb3 is an unidentified contaminant.
    Figure Legend Snippet: Purification of Drosophila NELF. ( A ) Nuclear extracts were prepared from transgenic fly embryos expressing FLAG-NELF subunits and fractionated using anti-FLAG Sepharose (Sigma). Eluates (10 uL) were analyzed by SDS-PAGE and Coomassie staining (lanes 2–4). Arrows indicate positions of the NELF subunits. Lane 1 (Control) shows 10 uL of eluate from a mock purification of nuclear extract from nontransgenic embryos. Asterisks denote proteins that bind nonspecifically to the FLAG column. ( B ) Western blot analysis of the FLAG NELF-D complex using NELF-A, NELF-B, NELF-D, and NELF-E antibodies. ( C ) Silver-stained gel with Pol II purified from Drosophila embryo nuclear extract and Coomassie blue-stained gel with DSIF purified from baculovirus. The prominent band migrating just above Rpb3 is an unidentified contaminant.

    Techniques Used: Purification, Transgenic Assay, Expressing, SDS Page, Staining, Western Blot

    36) Product Images from "Construction of New Ligation-Independent Cloning Vectors for the Expression and Purification of Recombinant Proteins in Silkworms Using BmNPV Bacmid System"

    Article Title: Construction of New Ligation-Independent Cloning Vectors for the Expression and Purification of Recombinant Proteins in Silkworms Using BmNPV Bacmid System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064007

    SDS-PAGE analysis of each purified KGDH subunit. The purified E1, E2, and E3 proteins were analyzed by SDS-PAGE with silver staining. Lane 1: E1 purified by Strep-Tactin agarose gel, lane 2: E3 purified by anti-FLAG M2 antibody agarose gel, lane 3: E2 purified by TALON affinity gel.
    Figure Legend Snippet: SDS-PAGE analysis of each purified KGDH subunit. The purified E1, E2, and E3 proteins were analyzed by SDS-PAGE with silver staining. Lane 1: E1 purified by Strep-Tactin agarose gel, lane 2: E3 purified by anti-FLAG M2 antibody agarose gel, lane 3: E2 purified by TALON affinity gel.

    Techniques Used: SDS Page, Purification, Silver Staining, Agarose Gel Electrophoresis

    37) Product Images from "TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1"

    Article Title: TRIM6 promotes colorectal cancer cells proliferation and response to thiostrepton by TIS21/FoxM1

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1504-5

    TRIM6 promoted TIS21 ubiquitination. a , b , Western blotting ( a ) and qRT-PCR ( b ) were used to detect TIS21 in HCT-8 and HCT116 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1, − 2) or control shRNA (shNC). c , HCT-8 cells were transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc (Vector) for 24 h, and exposed to 20 mM cycloheximide (CHX, Sigma-Aldrich). Cell lysate was prepared at 0, 3 and 6 h after exposure and subjected to western blotting analysis. d , HCT-8 cells were transfected with pCMV-Tag2-TIRM6 or pCMV-Tag2 vector for 24 h and then treated with MG132 (10 μM) or DMSO for 20 h. Western blotting was used to detect TIS21. e , Cell lysates from HCT-8 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1) or control shRNA (shNC) were IP with TIS21-Ab/control IgG and then immunoblotted for ubiquitin (Ub). f , Ubiquitination assay. The 293 T cells were transfected with plasmids expressing myc-TRIM6, His-ubiquitin and FLAG-TIS21 (WT, K5R, K51R or K150R). Cell lysates were incubated with nickelnitrilotriacetic acid beads and subjected to western blotting with anti-FLAG
    Figure Legend Snippet: TRIM6 promoted TIS21 ubiquitination. a , b , Western blotting ( a ) and qRT-PCR ( b ) were used to detect TIS21 in HCT-8 and HCT116 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1, − 2) or control shRNA (shNC). c , HCT-8 cells were transfected with pcDNA3.1-myc-TIRM6 or pcDNA3.1-myc (Vector) for 24 h, and exposed to 20 mM cycloheximide (CHX, Sigma-Aldrich). Cell lysate was prepared at 0, 3 and 6 h after exposure and subjected to western blotting analysis. d , HCT-8 cells were transfected with pCMV-Tag2-TIRM6 or pCMV-Tag2 vector for 24 h and then treated with MG132 (10 μM) or DMSO for 20 h. Western blotting was used to detect TIS21. e , Cell lysates from HCT-8 cells infected with lentivirus expressing TRIM6 shRNA (shTRIM6–1) or control shRNA (shNC) were IP with TIS21-Ab/control IgG and then immunoblotted for ubiquitin (Ub). f , Ubiquitination assay. The 293 T cells were transfected with plasmids expressing myc-TRIM6, His-ubiquitin and FLAG-TIS21 (WT, K5R, K51R or K150R). Cell lysates were incubated with nickelnitrilotriacetic acid beads and subjected to western blotting with anti-FLAG

    Techniques Used: Western Blot, Quantitative RT-PCR, Infection, Expressing, shRNA, Transfection, Plasmid Preparation, Ubiquitin Assay, Incubation

    TRIM6 interacted with TIS21 in CRC cells. a , pCMV-Tag2-TIRM6 or pCMV-Tag2 vector was transfected into 293 T cells, and 48 h later, cell lysates were prepared and subjected to immunoprecipitation (IP) experiments with anti-FLAG beads. After elusion with FLAG peptide, the immunoprecipitated protein complexes were resolved on SDS-PAGE, and stained with Coomassie Brilliant Blue. B, C, IP was carried out with TRIM6 antibody (TRIM6-Ab) /IgG ( b ) or TIS21 antibody (TIS21-Ab) /control IgG ( c ), and then western blotting was performed to analyze specific associations between TRIM6 and TIS21 in HCT-8 and HCT116 cells. D-E, GST pull-down assay. HCT-8 cells were lysed and incubated with GST, GST-tagged TRIM6 ( d ) and GST-tagged TIS21 ( e ) bound to glutathione beads, respectively. Proteins were detected as indicated. E, immunofluorescence staining of TRIM6 (Red) and TIS21 (Green) in HCT-8 and HCT116 cells. DAPI (blue) was used to label nuclei. Scale bar: 50 μm
    Figure Legend Snippet: TRIM6 interacted with TIS21 in CRC cells. a , pCMV-Tag2-TIRM6 or pCMV-Tag2 vector was transfected into 293 T cells, and 48 h later, cell lysates were prepared and subjected to immunoprecipitation (IP) experiments with anti-FLAG beads. After elusion with FLAG peptide, the immunoprecipitated protein complexes were resolved on SDS-PAGE, and stained with Coomassie Brilliant Blue. B, C, IP was carried out with TRIM6 antibody (TRIM6-Ab) /IgG ( b ) or TIS21 antibody (TIS21-Ab) /control IgG ( c ), and then western blotting was performed to analyze specific associations between TRIM6 and TIS21 in HCT-8 and HCT116 cells. D-E, GST pull-down assay. HCT-8 cells were lysed and incubated with GST, GST-tagged TRIM6 ( d ) and GST-tagged TIS21 ( e ) bound to glutathione beads, respectively. Proteins were detected as indicated. E, immunofluorescence staining of TRIM6 (Red) and TIS21 (Green) in HCT-8 and HCT116 cells. DAPI (blue) was used to label nuclei. Scale bar: 50 μm

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, SDS Page, Staining, Western Blot, Pull Down Assay, Incubation, Immunofluorescence

    38) Product Images from "Transcriptional Activation by MEIS1A in Response to Protein Kinase A Signaling Requires the Transducers of Regulated CREB Family of CREB Co-activators *"

    Article Title: Transcriptional Activation by MEIS1A in Response to Protein Kinase A Signaling Requires the Transducers of Regulated CREB Family of CREB Co-activators *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.005090

    MEIS1A interacts with TORC1 and TORC2. A , co-immunoprecipitation assay of FLAG-tagged TORC1 and untagged MEIS1A in transfected HEK293 cells. Anti-MEIS NT and anti-FLAG Western blot ( WB ) analyses were performed on FLAG-TORC1 immunoprecipitates ( IP ) prepared with anti-FLAG M2 affinity agarose. 10% input levels of MEIS1A and FLAG-TORC1 are indicated. B , upper panel , Western blot analysis of transfected and endogenous MEIS1A detected in immunoprecipitates of endogenous TORC2 from HEK293 cells. The experiment was performed in triplicate and the results of each assay are shown. Lower panel , anti-TORC2 Western blot analysis showing immunoprecipitated TORC2 by the anti-TORC2 antibody but not the control anti-GAL4 antibody. 10% input levels of MEIS1A and TORC2 are shown. C , a MEIS1A mutant lacking the C terminus fails to co-immunoprecipitate with TORC1. HEK293T cells were co-transfected with a FLAG-tagged TORC1 expression vector and a vector encoding either wild-type MEIS1A or a mutant lacking the TORC-responsive C terminus (MEIS1A-(Δ334–390)). On the second day following transfection, cells were treated with the proteasome inhibitor MG132 and cell lysates prepared 5 h later. Immunoprecipitation of TORC1 was performed with an anti-FLAG antibody, and the presence of MEIS1 proteins in the immunoprecipitates was subsequently assessed by Western blotting.
    Figure Legend Snippet: MEIS1A interacts with TORC1 and TORC2. A , co-immunoprecipitation assay of FLAG-tagged TORC1 and untagged MEIS1A in transfected HEK293 cells. Anti-MEIS NT and anti-FLAG Western blot ( WB ) analyses were performed on FLAG-TORC1 immunoprecipitates ( IP ) prepared with anti-FLAG M2 affinity agarose. 10% input levels of MEIS1A and FLAG-TORC1 are indicated. B , upper panel , Western blot analysis of transfected and endogenous MEIS1A detected in immunoprecipitates of endogenous TORC2 from HEK293 cells. The experiment was performed in triplicate and the results of each assay are shown. Lower panel , anti-TORC2 Western blot analysis showing immunoprecipitated TORC2 by the anti-TORC2 antibody but not the control anti-GAL4 antibody. 10% input levels of MEIS1A and TORC2 are shown. C , a MEIS1A mutant lacking the C terminus fails to co-immunoprecipitate with TORC1. HEK293T cells were co-transfected with a FLAG-tagged TORC1 expression vector and a vector encoding either wild-type MEIS1A or a mutant lacking the TORC-responsive C terminus (MEIS1A-(Δ334–390)). On the second day following transfection, cells were treated with the proteasome inhibitor MG132 and cell lysates prepared 5 h later. Immunoprecipitation of TORC1 was performed with an anti-FLAG antibody, and the presence of MEIS1 proteins in the immunoprecipitates was subsequently assessed by Western blotting.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Expressing, Plasmid Preparation

    A MEIS1 interaction domain maps to a coiled-coil region at the TORC1 N terminus. Upper panel , co-immunoprecipitation between untagged MEIS1A and full-length FLAG-tagged TORC1 ( Flag-TORC1 ) or its deletion derivatives in transfected HEK293 cells. MEIS1A proteins co-precipitated with FLAG-TORC1 derivatives prepared using anti-FLAG M2 affinity agarose were revealed by Western blot ( WB ) analysis with anti-MEIS NT antibody. The bottom two panels show inputs of MEIS1A and FLAG-TORC1 derivatives, respectively. Lower panel , schematic diagram of TORC1 constructs and their MEIS1A binding activities. WB , Western blot; IP , immunoprecipitation. The plus and minus signs below Binding correlate with the extent of binding to MEIS1A by the various TORC1 mutants.
    Figure Legend Snippet: A MEIS1 interaction domain maps to a coiled-coil region at the TORC1 N terminus. Upper panel , co-immunoprecipitation between untagged MEIS1A and full-length FLAG-tagged TORC1 ( Flag-TORC1 ) or its deletion derivatives in transfected HEK293 cells. MEIS1A proteins co-precipitated with FLAG-TORC1 derivatives prepared using anti-FLAG M2 affinity agarose were revealed by Western blot ( WB ) analysis with anti-MEIS NT antibody. The bottom two panels show inputs of MEIS1A and FLAG-TORC1 derivatives, respectively. Lower panel , schematic diagram of TORC1 constructs and their MEIS1A binding activities. WB , Western blot; IP , immunoprecipitation. The plus and minus signs below Binding correlate with the extent of binding to MEIS1A by the various TORC1 mutants.

    Techniques Used: Immunoprecipitation, Transfection, Western Blot, Construct, Binding Assay

    Knockdown of TORCs prevents PKA-mediated activation of the MEIS1A C terminus. A , upper panel , effect of TORC2 shRNA or non-silencing control ( CTRL ) shRNA on GAL-MEIS1A-(335–390) luciferase transcription augmented by TORC2. The indicated plasmids were co-transfected with the pML5xUAS reporter in HEK293 cells. Lower panel , knockdown of FLAG-TORC2 protein levels in TORC2 or control shRNA-treated cells was verified by immunoprecipitation with M2 beads followed by Western blot ( WB ) analysis with an anti-FLAG antibody. Cell extracts were probed for tubulin, confirming equivalent protein concentrations in each sample. FLAG-TORC2(Wobble) served as an RNA interference-resistant control. B , the role of endogenous TORC2 on transcriptional activation through the MEIS1A C terminus. Cells were transfected with the pML5xUAS reporter and expression vectors for either the GAL DBD or GAL-MEIS1A-(335–390), along with a PKA expression vector or empty plasmid. Transcriptional activation by PKA through the MEIS1A C terminus was abrogated by coexpression with the TORC2-specific shRNA but not the control shRNA. The experiment was conducted in triplicate. Error bars are S.D., and p signifies the results of the Student's t test applied to values for PKA-induced activity in the presence of control shRNA versus TORC2 shRNA. RLU , relative luciferase units.
    Figure Legend Snippet: Knockdown of TORCs prevents PKA-mediated activation of the MEIS1A C terminus. A , upper panel , effect of TORC2 shRNA or non-silencing control ( CTRL ) shRNA on GAL-MEIS1A-(335–390) luciferase transcription augmented by TORC2. The indicated plasmids were co-transfected with the pML5xUAS reporter in HEK293 cells. Lower panel , knockdown of FLAG-TORC2 protein levels in TORC2 or control shRNA-treated cells was verified by immunoprecipitation with M2 beads followed by Western blot ( WB ) analysis with an anti-FLAG antibody. Cell extracts were probed for tubulin, confirming equivalent protein concentrations in each sample. FLAG-TORC2(Wobble) served as an RNA interference-resistant control. B , the role of endogenous TORC2 on transcriptional activation through the MEIS1A C terminus. Cells were transfected with the pML5xUAS reporter and expression vectors for either the GAL DBD or GAL-MEIS1A-(335–390), along with a PKA expression vector or empty plasmid. Transcriptional activation by PKA through the MEIS1A C terminus was abrogated by coexpression with the TORC2-specific shRNA but not the control shRNA. The experiment was conducted in triplicate. Error bars are S.D., and p signifies the results of the Student's t test applied to values for PKA-induced activity in the presence of control shRNA versus TORC2 shRNA. RLU , relative luciferase units.

    Techniques Used: Activation Assay, shRNA, Luciferase, Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Activity Assay

    39) Product Images from "Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *"

    Article Title: Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.736801

    Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.
    Figure Legend Snippet: Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.

    Techniques Used: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Derivative Assay, Negative Control, Western Blot, Positive Control, Purification, Variant Assay

    Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .
    Figure Legend Snippet: Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .

    Techniques Used: Binding Assay, Activation Assay, FLAG-tag, Variant Assay, Fluorescence, Generated, Immunoprecipitation, SDS Page, Western Blot

    40) Product Images from "Comparison of Strong Cation Exchange and SDS/PAGE Fractionation for Analysis of Multi-Protein Complexes"

    Article Title: Comparison of Strong Cation Exchange and SDS/PAGE Fractionation for Analysis of Multi-Protein Complexes

    Journal: Journal of proteome research

    doi: 10.1021/pr100843x

    Schematic illustrating comparison of SDS-PAGE and SCX fractionation for the analysis of immunoprecipitated protein complexes (A) Immunoprecipitation was performed from HEK293T cells transfected with 3x-FLAG-Bmi-1 or 3x-FLAG-GATA3. Cells transfected with the vector alone was used as a negative control. Half of the eluate was separated on a 4–12% SDS-PAGE gel [Gel 2] and the proteins were stained with Coomassie Blue. The second half of the eluate was further divided into two halves; one half being loaded onto a 4–12% SDS-PAGE gel [Gel 1] and other digested with trypsin followed by separation using strong cation exchange [SCX] into 12 aliquots. Each lane of the SDS-PAGE gel was cut into 12 pieces, which were subjected to in-gel digestion and peptide extraction. The peptides present within the 12 SDS-PAGE bands and 12 SCX fractions were analyzed using identical RPLC-MS/MS conditions. (B) Eluate from the immuno-precipitate was separated onto a 4–12% SDS-PAGE gel and stained with a coomassie blue. The lane was cut into 12 bands and in-situ trypsinization and extraction of the peptides was performed. The peptides were analyzed using RPLC-MS/MS. Arrow heads show bands corresponding to the transfected proteins.
    Figure Legend Snippet: Schematic illustrating comparison of SDS-PAGE and SCX fractionation for the analysis of immunoprecipitated protein complexes (A) Immunoprecipitation was performed from HEK293T cells transfected with 3x-FLAG-Bmi-1 or 3x-FLAG-GATA3. Cells transfected with the vector alone was used as a negative control. Half of the eluate was separated on a 4–12% SDS-PAGE gel [Gel 2] and the proteins were stained with Coomassie Blue. The second half of the eluate was further divided into two halves; one half being loaded onto a 4–12% SDS-PAGE gel [Gel 1] and other digested with trypsin followed by separation using strong cation exchange [SCX] into 12 aliquots. Each lane of the SDS-PAGE gel was cut into 12 pieces, which were subjected to in-gel digestion and peptide extraction. The peptides present within the 12 SDS-PAGE bands and 12 SCX fractions were analyzed using identical RPLC-MS/MS conditions. (B) Eluate from the immuno-precipitate was separated onto a 4–12% SDS-PAGE gel and stained with a coomassie blue. The lane was cut into 12 bands and in-situ trypsinization and extraction of the peptides was performed. The peptides were analyzed using RPLC-MS/MS. Arrow heads show bands corresponding to the transfected proteins.

    Techniques Used: SDS Page, Fractionation, Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Staining, Mass Spectrometry, In Situ

    Related Articles

    Co-Immunoprecipitation Assay:

    Article Title: The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein
    Article Snippet: .. Beads were washed three times with Co-IP buffer, and bound proteins were eluted using 3×FLAG peptide (Sigma-Aldrich) at a concentration of 150 μg/ml in 100 μl Co-IP buffer. .. For other immunoprecipitations, magnetic beads (Pierce Crosslink Magnetic IP/Co-IP kit; Thermo Fisher Scientific) were coated with anti-N505 serum (5 μl serum per 25 μl beads) and cross-linked according to the manual.

    Concentration Assay:

    Article Title: The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein
    Article Snippet: .. Beads were washed three times with Co-IP buffer, and bound proteins were eluted using 3×FLAG peptide (Sigma-Aldrich) at a concentration of 150 μg/ml in 100 μl Co-IP buffer. .. For other immunoprecipitations, magnetic beads (Pierce Crosslink Magnetic IP/Co-IP kit; Thermo Fisher Scientific) were coated with anti-N505 serum (5 μl serum per 25 μl beads) and cross-linked according to the manual.

    Incubation:

    Article Title: TREM-1 multimerization is essential for its activation on monocytes and neutrophils
    Article Snippet: .. When indicated, cells were incubated with the clinical-stage TREM-1 inhibitory peptide LR12 at 25 µg/ml or cytochalasin D (Sigma-Aldrich) at 5 µg/ml. .. TREM-1 expression was assessed by staining with anti-hTREM-1-allophycocyanin (APC) or the corresponding isotype-APC antibodies (Miltenyi Biotec, Germany), and data were collected by flow cytometry (C6 Accuri, BD, USA).

    Article Title: Growth Differentiation Factor-15 Suppresses Maturation and Function of Dendritic Cells and Inhibits Tumor-Specific Immune Response
    Article Snippet: .. Next, the cells were incubated for 30 min after the addition of the T7 phage peptide library of human liver tumor cDNA (Novagen, USA). .. After the incubation, the cells were pelleted by centrifugation at 1500 rpm for 2 min. After cells were washed twice with Tris-buffer saline solution (TBS), resuspended in elution buffer and centrifuged, the supernatant was collected, and the iDCs were removed by centrifugation.

    other:

    Article Title: Emetine Promotes von Hippel-Lindau-Independent Degradation of Hypoxia-Inducible Factor-2α in Clear Cell Renal Carcinoma
    Article Snippet: Calpain inhibitor I [ N- acetyl-Leu-Leu-norleucinal (ALLN)], calpain inhibitor II [ N -acetyl- l -leucyl- l -leucyl- l -methioninal (ALLM)], calpastatin peptide, and the proteasome inhibitor N -benzoyloxycarbonyl ( Z )-Leu-Leu-leucinal (MG132) were purchased from Sigma-Aldrich (St. Louis, MO).

    Activity Assay:

    Article Title: Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors
    Article Snippet: .. Analysis of caspase-3 activity The activity of caspase-3, a main executioner protease involved in the apoptotic process, was determined by release of 7-amino-4-trifluoromethyl-coumarin (AFC) from a DEVD-AFC peptide substrate (Sigma). .. Cells (2×106 ), both control and samples exposed to S. aureus , with or without apoptotic stimuli were collected by centrifugation (200×g, 5 min, 4°C), washed with ice-cold PBS and resuspended in 100 µL of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS).

    Recombinant:

    Article Title: Functional Characterization of a Ficolin-mediated Complement Pathway in Amphioxus *
    Article Snippet: .. The recombinant BjFCN1 protein (without signal peptide) fused with TRX-His tag was expressed using pET32a vector (Novagen). .. GST-tagged recombinant BjMASP1/3-N protein (common N-terminal portion of BjMASP1 and 3 without signal peptide, CUB1-EGF-CUB2 domain) was expressed using pGEX 4T-2 vector (Promega).

    Plasmid Preparation:

    Article Title: Functional Characterization of a Ficolin-mediated Complement Pathway in Amphioxus *
    Article Snippet: .. The recombinant BjFCN1 protein (without signal peptide) fused with TRX-His tag was expressed using pET32a vector (Novagen). .. GST-tagged recombinant BjMASP1/3-N protein (common N-terminal portion of BjMASP1 and 3 without signal peptide, CUB1-EGF-CUB2 domain) was expressed using pGEX 4T-2 vector (Promega).

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  • 99
    Millipore 3x flag peptide
    The RNF213 RING domain can catalyze K6-dependent ubiquitin linkages. a , Schematic of RNF213 structure, illustrating sub-domains, and multiple sequence alignment showing closest relatives of RNF213 RING. Alignment was performed by searching the RNF213 RING domain sequence against the human Uniprot database, using the default settings in Blastp. The top 8 sequences were selected a and alignment diagram was generated using Jalview. b , Coomassie blue stain of GST-RING RNF213 variants purified from E. coli (left panel) and scheme of in vitro auto-ubiquitylation assay (right panel) c , Auto-ubiquitylation assays using GST-RING RNF213 and members of the UBE2D family of E2s. d , GST-RNF213 RING has impaired auto-ubiquitylation activity in the presence of K6R ubiquitin mutant, but not other ubiquitin K > R mutants. e , In vitro auto-ubiquitylation assays were performed using GST-RING RNF213 and a ubiquitin mutant with all lysines mutated (K0) or single add-back (single functional lysine) mutants. Note that GST-RING RNF213 auto-ubiquitylation activity requires K6 on ubiquitin to form poly-ubiquitin linkages. f, Immunoblot of single cell clone shows absence of RNF213 in HeLa Flp-In T-Rex KO cells generated by CRISPR/Cas9 technology. g , HeLa Flp-In T-Rex KO cells from 1f reconstituted with doxycycline-inducible 3xFL-RNF213 were induced for 36 hr, lysed, and immunoblotted for the indicated proteins. h , HeLa Flp-In T-Rex KO cells expressing <t>3x-Flag-RNF213</t> upon doxycycline induction were transfected with Control or UBE2D2 siRNAs, followed by an HA-UB (HA-tagged Ubiquitin) expression construct. Cells were harvested 36 hours after transfection, with MG132 (10 µM) and Chloroquine (50 µM) added for the last 3 hours, lysed, and immunoblotted for the indicated proteins. Each immunoblot ( c-h ) is a representative image of at least three independent experiments.
    3x Flag Peptide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore flag peptide
    SCoV2 PLpro binds to and de-ISGylates MDA5-2CARD. (a) Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (PDB: 6YVA). Key residues that mediate ‘site 1’ interaction (N156 and R166/E167) or ‘site 2’ interaction (F69) in PLpro, as well as its catalytically-active site (C111), are indicated. (b) ISGylation of GST-MDA5-2CARD in HEK293T cells that were co-transfected for 20 h with vector or V5-tagged SCoV2 PLpro WT or mutants, along with <t>FLAG-ISG15,</t> HA-Ube1L, and FLAG-UbcH8, determined by GST-PD and IB with anti-FLAG and anti-GST. WCLs were probed by IB with anti-V5, anti-HA, anti-FLAG, and anti-Actin. (c) Binding of HA-tagged MDA5 or RIG-I to V5-SCoV2-PLpro or FLAG-MeV-V (control) in transiently transfected HEK293T cells, determined by HA-PD and IB with anti-V5 or anti-FLAG, and anti-HA. WCLs were probed by IB with anti-V5 and anti-FLAG. (d) Oligomerization of FLAG-MDA5-2CARD in HEK293T cells that were co-transfected with vector, or V5-SCoV2 PLpro WT or C111A for 24 h, assessed by Native PAGE and IB with anti-FLAG. WCLs were further analyzed by <t>SDS-PAGE</t> and probed by IB with anti-FLAG, anti-V5 and anti-Actin. (e) ISGylation of GST-MDA5-2CARD in HEK293T cells that also expressed FLAG-ISG15, HA-Ube1L and FLAG-UbcH8, and were co-transfected for 40 h with vector or the indicated V5-tagged coronaviral PLpro, determined by GST-PD and IB with anti-FLAG, anti-V5, and anti-GST. Data are representative of at least two independent experiments.
    Flag Peptide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 336 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The RNF213 RING domain can catalyze K6-dependent ubiquitin linkages. a , Schematic of RNF213 structure, illustrating sub-domains, and multiple sequence alignment showing closest relatives of RNF213 RING. Alignment was performed by searching the RNF213 RING domain sequence against the human Uniprot database, using the default settings in Blastp. The top 8 sequences were selected a and alignment diagram was generated using Jalview. b , Coomassie blue stain of GST-RING RNF213 variants purified from E. coli (left panel) and scheme of in vitro auto-ubiquitylation assay (right panel) c , Auto-ubiquitylation assays using GST-RING RNF213 and members of the UBE2D family of E2s. d , GST-RNF213 RING has impaired auto-ubiquitylation activity in the presence of K6R ubiquitin mutant, but not other ubiquitin K > R mutants. e , In vitro auto-ubiquitylation assays were performed using GST-RING RNF213 and a ubiquitin mutant with all lysines mutated (K0) or single add-back (single functional lysine) mutants. Note that GST-RING RNF213 auto-ubiquitylation activity requires K6 on ubiquitin to form poly-ubiquitin linkages. f, Immunoblot of single cell clone shows absence of RNF213 in HeLa Flp-In T-Rex KO cells generated by CRISPR/Cas9 technology. g , HeLa Flp-In T-Rex KO cells from 1f reconstituted with doxycycline-inducible 3xFL-RNF213 were induced for 36 hr, lysed, and immunoblotted for the indicated proteins. h , HeLa Flp-In T-Rex KO cells expressing 3x-Flag-RNF213 upon doxycycline induction were transfected with Control or UBE2D2 siRNAs, followed by an HA-UB (HA-tagged Ubiquitin) expression construct. Cells were harvested 36 hours after transfection, with MG132 (10 µM) and Chloroquine (50 µM) added for the last 3 hours, lysed, and immunoblotted for the indicated proteins. Each immunoblot ( c-h ) is a representative image of at least three independent experiments.

    Journal: bioRxiv

    Article Title: Moyamoya Disease-Associated RNF213 Alleles Encode Dominant Negative Alleles That Globally Impair Ubiquitylation

    doi: 10.1101/2020.05.24.113795

    Figure Lengend Snippet: The RNF213 RING domain can catalyze K6-dependent ubiquitin linkages. a , Schematic of RNF213 structure, illustrating sub-domains, and multiple sequence alignment showing closest relatives of RNF213 RING. Alignment was performed by searching the RNF213 RING domain sequence against the human Uniprot database, using the default settings in Blastp. The top 8 sequences were selected a and alignment diagram was generated using Jalview. b , Coomassie blue stain of GST-RING RNF213 variants purified from E. coli (left panel) and scheme of in vitro auto-ubiquitylation assay (right panel) c , Auto-ubiquitylation assays using GST-RING RNF213 and members of the UBE2D family of E2s. d , GST-RNF213 RING has impaired auto-ubiquitylation activity in the presence of K6R ubiquitin mutant, but not other ubiquitin K > R mutants. e , In vitro auto-ubiquitylation assays were performed using GST-RING RNF213 and a ubiquitin mutant with all lysines mutated (K0) or single add-back (single functional lysine) mutants. Note that GST-RING RNF213 auto-ubiquitylation activity requires K6 on ubiquitin to form poly-ubiquitin linkages. f, Immunoblot of single cell clone shows absence of RNF213 in HeLa Flp-In T-Rex KO cells generated by CRISPR/Cas9 technology. g , HeLa Flp-In T-Rex KO cells from 1f reconstituted with doxycycline-inducible 3xFL-RNF213 were induced for 36 hr, lysed, and immunoblotted for the indicated proteins. h , HeLa Flp-In T-Rex KO cells expressing 3x-Flag-RNF213 upon doxycycline induction were transfected with Control or UBE2D2 siRNAs, followed by an HA-UB (HA-tagged Ubiquitin) expression construct. Cells were harvested 36 hours after transfection, with MG132 (10 µM) and Chloroquine (50 µM) added for the last 3 hours, lysed, and immunoblotted for the indicated proteins. Each immunoblot ( c-h ) is a representative image of at least three independent experiments.

    Article Snippet: Beads were collected at bottom of the tube using a magnetic stand, supernatants were discarded, and immunoprecipitates were washed 5 times in lysis buffer followed by elution with 50µL of 100ng/µL 3x-Flag peptide.

    Techniques: Sequencing, Generated, Staining, Purification, In Vitro, Ubiquitin Assay, Activity Assay, Mutagenesis, Functional Assay, CRISPR, Expressing, Transfection, Construct

    MMD SNPs do not affect AAA+ ATPase activity of RNF213 a , Schematic showing RNF213 WT and positions of AAA+ ATPase (E2488Q, E2845Q), RING (I3999A) and MMD-associated (D4013N, H4014N, K4732T, R4810K) mutants. b , HeLa Flp-In T-Rex KO cells were transfected with 3x Flag RNF213 WT , 3x Flag RNF213 E2488Q, E2845Q , 3x-Flag-RNF213 I3999A , 3x-Flag-RNF213 D4013N , 3x-Flag-RNF213 H4014N , 3x-Flag-RNF213 K4732T or 3x-Flag-RNF213 R4810K . Thirty-six hours post-transfection, cells were harvested and lysates were subjected to immunoprecipitation using anti-Flag antibody. Purified RNF213 variants were detected by SDS-PAGE and Coomassie blue staining. BSA was loaded to aid in quantification of RNF213. c , ATPase activity of purified RNF213 variants from Fig. 2b . Error bars represent means ± SD of three independent experiments, each with samples in triplicate. Statistical significance was evaluated by two-way ANOVA, followed by Dunnett test.

    Journal: bioRxiv

    Article Title: Moyamoya Disease-Associated RNF213 Alleles Encode Dominant Negative Alleles That Globally Impair Ubiquitylation

    doi: 10.1101/2020.05.24.113795

    Figure Lengend Snippet: MMD SNPs do not affect AAA+ ATPase activity of RNF213 a , Schematic showing RNF213 WT and positions of AAA+ ATPase (E2488Q, E2845Q), RING (I3999A) and MMD-associated (D4013N, H4014N, K4732T, R4810K) mutants. b , HeLa Flp-In T-Rex KO cells were transfected with 3x Flag RNF213 WT , 3x Flag RNF213 E2488Q, E2845Q , 3x-Flag-RNF213 I3999A , 3x-Flag-RNF213 D4013N , 3x-Flag-RNF213 H4014N , 3x-Flag-RNF213 K4732T or 3x-Flag-RNF213 R4810K . Thirty-six hours post-transfection, cells were harvested and lysates were subjected to immunoprecipitation using anti-Flag antibody. Purified RNF213 variants were detected by SDS-PAGE and Coomassie blue staining. BSA was loaded to aid in quantification of RNF213. c , ATPase activity of purified RNF213 variants from Fig. 2b . Error bars represent means ± SD of three independent experiments, each with samples in triplicate. Statistical significance was evaluated by two-way ANOVA, followed by Dunnett test.

    Article Snippet: Beads were collected at bottom of the tube using a magnetic stand, supernatants were discarded, and immunoprecipitates were washed 5 times in lysis buffer followed by elution with 50µL of 100ng/µL 3x-Flag peptide.

    Techniques: Activity Assay, Transfection, Immunoprecipitation, Purification, SDS Page, Staining

    SCoV2 PLpro binds to and de-ISGylates MDA5-2CARD. (a) Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (PDB: 6YVA). Key residues that mediate ‘site 1’ interaction (N156 and R166/E167) or ‘site 2’ interaction (F69) in PLpro, as well as its catalytically-active site (C111), are indicated. (b) ISGylation of GST-MDA5-2CARD in HEK293T cells that were co-transfected for 20 h with vector or V5-tagged SCoV2 PLpro WT or mutants, along with FLAG-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by GST-PD and IB with anti-FLAG and anti-GST. WCLs were probed by IB with anti-V5, anti-HA, anti-FLAG, and anti-Actin. (c) Binding of HA-tagged MDA5 or RIG-I to V5-SCoV2-PLpro or FLAG-MeV-V (control) in transiently transfected HEK293T cells, determined by HA-PD and IB with anti-V5 or anti-FLAG, and anti-HA. WCLs were probed by IB with anti-V5 and anti-FLAG. (d) Oligomerization of FLAG-MDA5-2CARD in HEK293T cells that were co-transfected with vector, or V5-SCoV2 PLpro WT or C111A for 24 h, assessed by Native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-V5 and anti-Actin. (e) ISGylation of GST-MDA5-2CARD in HEK293T cells that also expressed FLAG-ISG15, HA-Ube1L and FLAG-UbcH8, and were co-transfected for 40 h with vector or the indicated V5-tagged coronaviral PLpro, determined by GST-PD and IB with anti-FLAG, anti-V5, and anti-GST. Data are representative of at least two independent experiments.

    Journal: bioRxiv

    Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

    doi: 10.1101/2020.10.26.356048

    Figure Lengend Snippet: SCoV2 PLpro binds to and de-ISGylates MDA5-2CARD. (a) Ribbon representation of the crystal structure of the SCoV2 PLpro: ISG15 complex (PDB: 6YVA). Key residues that mediate ‘site 1’ interaction (N156 and R166/E167) or ‘site 2’ interaction (F69) in PLpro, as well as its catalytically-active site (C111), are indicated. (b) ISGylation of GST-MDA5-2CARD in HEK293T cells that were co-transfected for 20 h with vector or V5-tagged SCoV2 PLpro WT or mutants, along with FLAG-ISG15, HA-Ube1L, and FLAG-UbcH8, determined by GST-PD and IB with anti-FLAG and anti-GST. WCLs were probed by IB with anti-V5, anti-HA, anti-FLAG, and anti-Actin. (c) Binding of HA-tagged MDA5 or RIG-I to V5-SCoV2-PLpro or FLAG-MeV-V (control) in transiently transfected HEK293T cells, determined by HA-PD and IB with anti-V5 or anti-FLAG, and anti-HA. WCLs were probed by IB with anti-V5 and anti-FLAG. (d) Oligomerization of FLAG-MDA5-2CARD in HEK293T cells that were co-transfected with vector, or V5-SCoV2 PLpro WT or C111A for 24 h, assessed by Native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-V5 and anti-Actin. (e) ISGylation of GST-MDA5-2CARD in HEK293T cells that also expressed FLAG-ISG15, HA-Ube1L and FLAG-UbcH8, and were co-transfected for 40 h with vector or the indicated V5-tagged coronaviral PLpro, determined by GST-PD and IB with anti-FLAG, anti-V5, and anti-GST. Data are representative of at least two independent experiments.

    Article Snippet: The beads were extensively washed with NP-40 buffer and proteins eluted by heating in 1× Laemmli SDS sample buffer at 95°C for 5 min or by competition with FLAG peptide (Millipore) 4°C for 4 h. For endogenous MDA5 immunoprecipitation, NHLFs were stimulated with poly(I:C) (HMW)/LyoVec (0.1 μg/mL) or infected with DENV or ZIKV at the indicated MOI for 40 h. Cell lysates were precleared with Protein G Dynabeads (Invitrogen) at 4°C for 2 h and then incubated with Protein G Dynabeads conjugated with the anti-MDA5 antibody or an IgG1 isotype control (G3A1; CST) at 4°C for 4 h. The beads were washed four times with RIPA buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) deoxycholic acid, 0.01% (w/v) SDS] and protein eluted in 1× Laemmli SDS sample buffer.

    Techniques: Transfection, Plasmid Preparation, Binding Assay, Clear Native PAGE, SDS Page

    CARD ISGylation is essential for formation of higher-order MDA5 assemblies. ( a , b) Cytosol-mitochondria fractionation of WCLs from NHLFs that were transfected for 30 h with non-targeting control siRNA (si.C) or ISG15-specific siRNA (si.ISG15) and then mock-treated or transfected with EMCV-RNA (0.4 μg/mL) (a) or RABVLe (1 pmol/mL) (b) for 16 h. IB was performed with anti-MDA5 (a), anti-RIG-I (b), anti-ISG15 and anti-Actin (a, b). α-Tubulin and MAVS served as purity markers for the cytosolic and mitochondrial fraction, respectively (a, b). (c) Endogenous MDA5 oligomerization in WT and Isg15 −/− MEFs that were transfected with EMCV-RNA (0.5 μg/mL) for 16 h, assessed by SDD-AGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (d) Oligomerization of FLAG-MDA5-2CARD in HEK293T cells that were transfected with the indicated siRNAs together with or without HA-Ube1L and FLAG-UbcH8 for 48 h, determined by native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-HA, anti-ISG15, and anti-Actin. (e) Oligomerization of FLAG-MDA5 WT and K23R/K43R in transiently transfected MDA5 KO HEK293 cells, assessed by SDD-AGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and IB with anti-FLAG and anti-Actin. (f) Oligomerization of FLAG-tagged MDA5 WT and mutants in transiently transfected MDA5 KO HEK293 cells, assessed by native PAGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (g) IFN-β-luciferase reporter activity in MDA5 KO HEK293 cells that were transfected for 24 h with either empty vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f). *** p

    Journal: bioRxiv

    Article Title: ISG15-dependent Activation of the RNA Sensor MDA5 and its Antagonism by the SARS-CoV-2 papain-like protease

    doi: 10.1101/2020.10.26.356048

    Figure Lengend Snippet: CARD ISGylation is essential for formation of higher-order MDA5 assemblies. ( a , b) Cytosol-mitochondria fractionation of WCLs from NHLFs that were transfected for 30 h with non-targeting control siRNA (si.C) or ISG15-specific siRNA (si.ISG15) and then mock-treated or transfected with EMCV-RNA (0.4 μg/mL) (a) or RABVLe (1 pmol/mL) (b) for 16 h. IB was performed with anti-MDA5 (a), anti-RIG-I (b), anti-ISG15 and anti-Actin (a, b). α-Tubulin and MAVS served as purity markers for the cytosolic and mitochondrial fraction, respectively (a, b). (c) Endogenous MDA5 oligomerization in WT and Isg15 −/− MEFs that were transfected with EMCV-RNA (0.5 μg/mL) for 16 h, assessed by SDD-AGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (d) Oligomerization of FLAG-MDA5-2CARD in HEK293T cells that were transfected with the indicated siRNAs together with or without HA-Ube1L and FLAG-UbcH8 for 48 h, determined by native PAGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-FLAG, anti-HA, anti-ISG15, and anti-Actin. (e) Oligomerization of FLAG-MDA5 WT and K23R/K43R in transiently transfected MDA5 KO HEK293 cells, assessed by SDD-AGE and IB with anti-FLAG. WCLs were further analyzed by SDS-PAGE and IB with anti-FLAG and anti-Actin. (f) Oligomerization of FLAG-tagged MDA5 WT and mutants in transiently transfected MDA5 KO HEK293 cells, assessed by native PAGE and IB with anti-MDA5. WCLs were further analyzed by SDS-PAGE and probed by IB with anti-MDA5 and anti-Actin. (g) IFN-β-luciferase reporter activity in MDA5 KO HEK293 cells that were transfected for 24 h with either empty vector, or FLAG-tagged MDA5 WT or mutants. Luciferase activity is presented as fold induction relative to the values for vector-transfected cells, set to 1. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates in f). *** p

    Article Snippet: The beads were extensively washed with NP-40 buffer and proteins eluted by heating in 1× Laemmli SDS sample buffer at 95°C for 5 min or by competition with FLAG peptide (Millipore) 4°C for 4 h. For endogenous MDA5 immunoprecipitation, NHLFs were stimulated with poly(I:C) (HMW)/LyoVec (0.1 μg/mL) or infected with DENV or ZIKV at the indicated MOI for 40 h. Cell lysates were precleared with Protein G Dynabeads (Invitrogen) at 4°C for 2 h and then incubated with Protein G Dynabeads conjugated with the anti-MDA5 antibody or an IgG1 isotype control (G3A1; CST) at 4°C for 4 h. The beads were washed four times with RIPA buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) deoxycholic acid, 0.01% (w/v) SDS] and protein eluted in 1× Laemmli SDS sample buffer.

    Techniques: Fractionation, Transfection, SDS Page, Clear Native PAGE, Luciferase, Activity Assay, Plasmid Preparation

    Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *

    doi: 10.1074/jbc.M116.736801

    Figure Lengend Snippet: Neutralizing effect of mAb 286, mapping of its binding site, and analysis of binding to VEGF-D variants with mutated residues in N-terminal α-helix. A , the capacity of mAb 286 to block binding and cross-linking, by VEGF-DΔNΔC, of chimeric receptors containing VEGFR-2 ( left ) or VEGFR-3 ( right ) extracellular domains was assessed in bioassays (see “Experimental Procedures”). Also included were neutralizing mAb VD1, which binds loop 2 of VEGF-DΔNΔC, and mAb VD4, which binds, but does not neutralize, VEGF-DΔNΔC ( 39 ). B , peptide-based mapping of the mAb 286 binding site in VEGF-DΔNΔC by ELISA (see “Experimental Procedures”). The ratio of signal to background for the interaction of mAb 286 with immobilized peptides is shown on the y axis of the graph, and the x axis indicates the identifier numbers of peptides. Top box above the graph , amino acid sequence for the VEGF homology domain of human VEGF-D; N-terminal residue (phenylalanine) is number 89, and the C-terminal residue (arginine) is 205. Bottom box above the graph , examples of peptides used in mapping (mAb 286 binding site is in a rectangle ). The FLAG sequence is shown in boldface type in peptide 36, which lacks the VEGF-D-derived sequence, and was the negative control. C , detection of VEGF-DΔNΔC variants by Western blotting under reducing and denaturing conditions using mAb 286 ( top ) or M2 anti-FLAG mAb as a positive control ( bottom ). Each well contained 30 ng of purified protein. VEGF-D , VEGF-DΔNΔC; variants of this protein each have one residue mutated to alanine, as indicated. Positions of molecular mass markers (in kDa) are shown to the left . The histogram under the blots shows intensities of bands for VEGF-D variants (mean ± S.D.) relative to the intensity of the band for VEGF-DΔNΔC, as determined from Western blots with mAb 286. D , analysis of mAb 286 binding to VEGF-DΔNΔC variants by ELISA. M2 was used for capture and mAb 286 for detection; the y axis shows binding of variant proteins compared with VEGF-DΔNΔC (the latter defined as 100% binding), and the x axis lists VEGF-D variants. Equal amounts of VEGF-DΔNΔC and variants were used. For A , B , and D , assays were conducted three times. Columns , mean; error bars , S.D.

    Article Snippet: Equal volumes of conditioned medium containing VEGF-DΔNΔC variants that were not tagged with the FLAG peptide were concentrated to the same final volume and buffer-exchanged into PBS using an Amicon size exclusion centrifugal filter with a 10 kDa nominal molecular mass limit (Millipore, Billerica, MA).

    Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Derivative Assay, Negative Control, Western Blot, Positive Control, Purification, Variant Assay

    Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .

    Journal: The Journal of Biological Chemistry

    Article Title: Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D *

    doi: 10.1074/jbc.M116.736801

    Figure Lengend Snippet: Receptor binding and activation by untagged VEGF-D variants. A , bioassays for binding and cross-linking of extracellular domains of VEGFR-2 ( left ) and VEGFR-3 ( right ) with altered versions of VEGF-DΔNΔC, Y94A, K100A, and I102A lacking FLAG tag. The same amount of each VEGF-DΔNΔC variant was used. Results are expressed as a percentage of fluorescence units generated relative to untagged VEGF-DΔNΔC ( y axis). VEGF-D , untagged form of VEGF-DΔNΔC. Assays were conducted three times. Columns , mean; error bars , S.D. *, statistically significant differences as assessed by one-way analysis of variance with Tukey's post hoc test. B , adult LECs were stimulated with matched quantities of untagged variants or left unstimulated ( No GF ). Lysates were immunoprecipitated ( IP ) with antibody against VEGFR-2 ( left ) or VEGFR-3 ( right ) and analyzed by reducing SDS-PAGE and Western blotting with antibody against phosphotyrosine ( pY ) to assess receptor activation ( top blots ) or with antibody against VEGFR-2 ( bottom left blot ) or VEGFR-3 ( bottom right blot ) to confirm the presence of each receptor. Sizes of molecular mass markers (in kDa) are shown to the left of the panels .

    Article Snippet: Equal volumes of conditioned medium containing VEGF-DΔNΔC variants that were not tagged with the FLAG peptide were concentrated to the same final volume and buffer-exchanged into PBS using an Amicon size exclusion centrifugal filter with a 10 kDa nominal molecular mass limit (Millipore, Billerica, MA).

    Techniques: Binding Assay, Activation Assay, FLAG-tag, Variant Assay, Fluorescence, Generated, Immunoprecipitation, SDS Page, Western Blot