monoclonal anti flag m2 antibody  (Millipore)

 
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    Name:
    Monoclonal ANTI FLAG M2 antibody
    Description:
    Anti Flag M2 antibody is used for the detection of Flag fusion proteins This monoclonal antibody is produced in mouse and recognizes the FLAG sequence at the N terminus Met N terminus and C terminus The antibody is also able to recognize FLAG at an internal site M2 unlike M1 antibody is not Calcium dependent F1804 is an affinity purified FLAG M2 antibody increasing sensitivity in most applications Method of purification Protein A
    Catalog Number:
    F3165
    Price:
    None
    Applications:
    Antibody is recommended for use in immunoblotting, immunoprecipitation, immunocytochemistry, immunofluorescence, ELISA, electron microscopy, flow cytometry and supershift assays.Browse additional application references in our FLAG(R) Literature portal.
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    Structured Review

    Millipore monoclonal anti flag m2 antibody
    HIGM2 mutations in AID disrupt its interactions with ROD1. a are shown at the exact residue positions. b The mutation residues are highly conserved. Amino acids from 139 to 153 were aligned. D.r. Zebrafish, X.l. frog, G.g. chicken, H.s. human, M.m. mouse. c The interacting surface between human AID and ROD1 modeled by PRISM. The mutations are marked in red. d His-ROD1 pulled-down GST-AID and some variant proteins, but not 147X variant. e Rescue of IgG1 CSR in AID −/− splenic B cells by transducing <t>Flag-tagged</t> AID or AID 147X mutant. Percentage of IgG1 CSR is the ratio between IgG1 + /GFP + cells and total GFP + cells. f <t>Anti-ROD1</t> immunoprecipitates from retrovirally transduced AID −/− B cells with either Flag-tagged AID or AID 147X mutant. g ChIP-qPCR analysis of AID and AID 147X mutant occupancy at S-γ1 and non- Ig targets in reconstituted AID −/− B cells as shown in f . S-γ3 and Cdc42 served as AID non-target controls. * P
    Anti Flag M2 antibody is used for the detection of Flag fusion proteins This monoclonal antibody is produced in mouse and recognizes the FLAG sequence at the N terminus Met N terminus and C terminus The antibody is also able to recognize FLAG at an internal site M2 unlike M1 antibody is not Calcium dependent F1804 is an affinity purified FLAG M2 antibody increasing sensitivity in most applications Method of purification Protein A
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes"

    Article Title: The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes

    Journal: Cell Research

    doi: 10.1038/s41422-018-0076-9

    HIGM2 mutations in AID disrupt its interactions with ROD1. a are shown at the exact residue positions. b The mutation residues are highly conserved. Amino acids from 139 to 153 were aligned. D.r. Zebrafish, X.l. frog, G.g. chicken, H.s. human, M.m. mouse. c The interacting surface between human AID and ROD1 modeled by PRISM. The mutations are marked in red. d His-ROD1 pulled-down GST-AID and some variant proteins, but not 147X variant. e Rescue of IgG1 CSR in AID −/− splenic B cells by transducing Flag-tagged AID or AID 147X mutant. Percentage of IgG1 CSR is the ratio between IgG1 + /GFP + cells and total GFP + cells. f Anti-ROD1 immunoprecipitates from retrovirally transduced AID −/− B cells with either Flag-tagged AID or AID 147X mutant. g ChIP-qPCR analysis of AID and AID 147X mutant occupancy at S-γ1 and non- Ig targets in reconstituted AID −/− B cells as shown in f . S-γ3 and Cdc42 served as AID non-target controls. * P
    Figure Legend Snippet: HIGM2 mutations in AID disrupt its interactions with ROD1. a are shown at the exact residue positions. b The mutation residues are highly conserved. Amino acids from 139 to 153 were aligned. D.r. Zebrafish, X.l. frog, G.g. chicken, H.s. human, M.m. mouse. c The interacting surface between human AID and ROD1 modeled by PRISM. The mutations are marked in red. d His-ROD1 pulled-down GST-AID and some variant proteins, but not 147X variant. e Rescue of IgG1 CSR in AID −/− splenic B cells by transducing Flag-tagged AID or AID 147X mutant. Percentage of IgG1 CSR is the ratio between IgG1 + /GFP + cells and total GFP + cells. f Anti-ROD1 immunoprecipitates from retrovirally transduced AID −/− B cells with either Flag-tagged AID or AID 147X mutant. g ChIP-qPCR analysis of AID and AID 147X mutant occupancy at S-γ1 and non- Ig targets in reconstituted AID −/− B cells as shown in f . S-γ3 and Cdc42 served as AID non-target controls. * P

    Techniques Used: Mutagenesis, Variant Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    2) Product Images from "Inhibition of HIV-1 Tat-Mediated Transcription by a Coumarin Derivative, BPRHIV001, through the Akt Pathway ▿"

    Article Title: Inhibition of HIV-1 Tat-Mediated Transcription by a Coumarin Derivative, BPRHIV001, through the Akt Pathway ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00175-11

    The formation of Tat/P-TEFb complex was not interrupted by BPRHIV001. (A) Unaffected Tat protein levels in the presence of BPRHIV001. Tat protein expression in the presence of different concentrations of BPRHIV001 was determined by Western blotting using the anti-Flag antibody. The relative expression of Tat normalized by β-actin was shown individually. (B) Unaltered amount of TAR RNA in the presence of BPRHIV001. RT-PCR was performed to determine the amount of TAR in the presence of BPRHIV001. The relative amount of TAR normalized by the amount of transfected pGL2-LTR DNA and 23S rRNA is shown individually. (C) No disruption of Tat/P-TEFb complex assembly in the presence of BPRHIV001. Coimmunoprecipitation was performed using cell lysates prepared from Tat-transfected cells treated with BPRHIV001 or DMSO alone. Anti-CDK9 polyclonal antibodies were used to pull down the protein complexes, which were further separated by electrophoresis. The amount of individual proteins was determined by Western blotting using specific antibodies. The relative amount of individual protein in the immunoprecipitated (IP) Tat/P-TEFb complex was normalized by CDK9 and is shown below the gel image.
    Figure Legend Snippet: The formation of Tat/P-TEFb complex was not interrupted by BPRHIV001. (A) Unaffected Tat protein levels in the presence of BPRHIV001. Tat protein expression in the presence of different concentrations of BPRHIV001 was determined by Western blotting using the anti-Flag antibody. The relative expression of Tat normalized by β-actin was shown individually. (B) Unaltered amount of TAR RNA in the presence of BPRHIV001. RT-PCR was performed to determine the amount of TAR in the presence of BPRHIV001. The relative amount of TAR normalized by the amount of transfected pGL2-LTR DNA and 23S rRNA is shown individually. (C) No disruption of Tat/P-TEFb complex assembly in the presence of BPRHIV001. Coimmunoprecipitation was performed using cell lysates prepared from Tat-transfected cells treated with BPRHIV001 or DMSO alone. Anti-CDK9 polyclonal antibodies were used to pull down the protein complexes, which were further separated by electrophoresis. The amount of individual proteins was determined by Western blotting using specific antibodies. The relative amount of individual protein in the immunoprecipitated (IP) Tat/P-TEFb complex was normalized by CDK9 and is shown below the gel image.

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Electrophoresis, Immunoprecipitation

    3) Product Images from "MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells"

    Article Title: MEG8 long noncoding RNA contributes to epigenetic progression of the epithelial-mesenchymal transition of lung and pancreatic cancer cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.004006

    MEG8 could mainly interact with EZH2 and associate with the specific regulatory regions of miR-34a and miR-203 genes for transcriptional regulation. A and B , interaction of overexpressed MEG3 or MEG8 lncRNA with JARID2 or EZH2 protein. A549 cells were infected with the various combinations (as indicated) of retroviruses expressing MEG3 , MEG8 , and FLAG-tagged JARID2 . The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG; C ), anti-EZH2 antibody ( E ), or anti-FLAG antibody ( F ), and the coprecipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 ( A ) or MEG8 ( B ) in the precipitates. n.d. means not detected. p values are based on one-way ANOVA with Bonferroni post-test (*, p
    Figure Legend Snippet: MEG8 could mainly interact with EZH2 and associate with the specific regulatory regions of miR-34a and miR-203 genes for transcriptional regulation. A and B , interaction of overexpressed MEG3 or MEG8 lncRNA with JARID2 or EZH2 protein. A549 cells were infected with the various combinations (as indicated) of retroviruses expressing MEG3 , MEG8 , and FLAG-tagged JARID2 . The cross-linked cell lysates were immunoprecipitated with control antibody (mouse IgG; C ), anti-EZH2 antibody ( E ), or anti-FLAG antibody ( F ), and the coprecipitated RNA was transcribed to cDNA. QPCR was performed to detect the enrichment of MEG3 ( A ) or MEG8 ( B ) in the precipitates. n.d. means not detected. p values are based on one-way ANOVA with Bonferroni post-test (*, p

    Techniques Used: Infection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

    4) Product Images from "The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators"

    Article Title: The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000678

    BRD3 binds to a short motif in the N-terminal disordered region of CHD4. A, human CHD4 constructs used in this study. Domains with known structures are indicated. The presence or absence of an interaction with BRD3 is indicated by + or −, respectively. CD = chromodomain. B, BRD3 coimmunoprecipitates with the N-terminal third of CHD4. BRD3-HA (full-length, L or BD ) and CHD4-FLAG (full-length, NT , M , or CT ) constructs were coexpressed in HEK293 cells and applied to anti-FLAG-agarose beads. Western blottings using anti-HA or anti-FLAG antibodies are shown. C, CHD4(265–310) ( fragment Ne ) binds BRD3. Bacterially expressed CHD4 fragments immobilized on GSH beads were used to pull down mammalian-expressed HA-BRD3-L. Each Western blotting was visualized using α-HA and α-GST antibodies. PD = pulldown; FT = flow-through; * = unidentified band.
    Figure Legend Snippet: BRD3 binds to a short motif in the N-terminal disordered region of CHD4. A, human CHD4 constructs used in this study. Domains with known structures are indicated. The presence or absence of an interaction with BRD3 is indicated by + or −, respectively. CD = chromodomain. B, BRD3 coimmunoprecipitates with the N-terminal third of CHD4. BRD3-HA (full-length, L or BD ) and CHD4-FLAG (full-length, NT , M , or CT ) constructs were coexpressed in HEK293 cells and applied to anti-FLAG-agarose beads. Western blottings using anti-HA or anti-FLAG antibodies are shown. C, CHD4(265–310) ( fragment Ne ) binds BRD3. Bacterially expressed CHD4 fragments immobilized on GSH beads were used to pull down mammalian-expressed HA-BRD3-L. Each Western blotting was visualized using α-HA and α-GST antibodies. PD = pulldown; FT = flow-through; * = unidentified band.

    Techniques Used: Construct, Western Blot, Flow Cytometry

    5) Product Images from "The ATP binding site of the chromatin remodeling homolog Lsh is required for nucleosome density and de novo DNA methylation at repeat sequences"

    Article Title: The ATP binding site of the chromatin remodeling homolog Lsh is required for nucleosome density and de novo DNA methylation at repeat sequences

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1371

    Re-induction of Lsh in KO ES cell restores DNA methylation during ES differentiation. ( A ) The regions analyzed by bisulfite sequencing in IAP LTR (5′ long terminal repeats), minor satellite sequence and Line1 elements are shown in a schematic graph. ( B ) Bisulfite sequencing analysis of the IAP elements, minor satellite sequences and Line1 elements in RA-treated WT ES cells (WT), KO ES cells (KO) and KO ES cells re-expressing full-length Lsh protein (KO+Lsh). A detailed RA differentiation protocol is shown in Supplementary Figure S 2B . The black filled circles represent CG methylation, while the white open circles indicate no methylation at specific CpG sites. Gaps in the methylation profiles indicate mutated or missing CpG sites. The percentage of methylated CpGs is shown below each group of clones. Each datum represents one representative experiment of two to four independent experiments (see Figure 1E ). ( C ) Western analysis for detection of flag-tagged Lsh protein. Undifferentiated KO ES cells transfected with Lsh expression vector (1), WT ES cells (2), WT ES cells re-expressing full-length wild-type Lsh (3) and KO ES cells (4). ( D ) Immune fluorescence analysis for detection of the nuclear location of Lsh in undifferentiated KO ES cells expressing full-length wild-type Lsh (KO+Lsh) using an anti-Lsh antibody or the antibody against the flag epitope. WT ES cells were used as positive control, KO ES cells served as negative control. Oct4 antibodies were used for detection of the pluripotency marker Oct4. Nuclei were stained with DAPI. ( E ) Bar graph representing CG methylation level in RA-treated WT ES cells (WT), KO ES cells (KO) and KO ES cells re-expressing full-length wild-type Lsh protein (KO+Lsh) at IAP sequences, minor satellite sequences and Line1 elements. The graph represents mean ± SD from four independent experiments (IAP, Line1) or two independent experiments (minor satellite), each including at least 10 sequenced clones. SD: standard deviation. * P
    Figure Legend Snippet: Re-induction of Lsh in KO ES cell restores DNA methylation during ES differentiation. ( A ) The regions analyzed by bisulfite sequencing in IAP LTR (5′ long terminal repeats), minor satellite sequence and Line1 elements are shown in a schematic graph. ( B ) Bisulfite sequencing analysis of the IAP elements, minor satellite sequences and Line1 elements in RA-treated WT ES cells (WT), KO ES cells (KO) and KO ES cells re-expressing full-length Lsh protein (KO+Lsh). A detailed RA differentiation protocol is shown in Supplementary Figure S 2B . The black filled circles represent CG methylation, while the white open circles indicate no methylation at specific CpG sites. Gaps in the methylation profiles indicate mutated or missing CpG sites. The percentage of methylated CpGs is shown below each group of clones. Each datum represents one representative experiment of two to four independent experiments (see Figure 1E ). ( C ) Western analysis for detection of flag-tagged Lsh protein. Undifferentiated KO ES cells transfected with Lsh expression vector (1), WT ES cells (2), WT ES cells re-expressing full-length wild-type Lsh (3) and KO ES cells (4). ( D ) Immune fluorescence analysis for detection of the nuclear location of Lsh in undifferentiated KO ES cells expressing full-length wild-type Lsh (KO+Lsh) using an anti-Lsh antibody or the antibody against the flag epitope. WT ES cells were used as positive control, KO ES cells served as negative control. Oct4 antibodies were used for detection of the pluripotency marker Oct4. Nuclei were stained with DAPI. ( E ) Bar graph representing CG methylation level in RA-treated WT ES cells (WT), KO ES cells (KO) and KO ES cells re-expressing full-length wild-type Lsh protein (KO+Lsh) at IAP sequences, minor satellite sequences and Line1 elements. The graph represents mean ± SD from four independent experiments (IAP, Line1) or two independent experiments (minor satellite), each including at least 10 sequenced clones. SD: standard deviation. * P

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Sequencing, Expressing, Methylation, Clone Assay, Western Blot, Transfection, Plasmid Preparation, Fluorescence, FLAG-tag, Positive Control, Negative Control, Marker, Staining, Standard Deviation

    6) Product Images from "TBL1 is required for the mesenchymal phenotype of transformed breast cancer cells"

    Article Title: TBL1 is required for the mesenchymal phenotype of transformed breast cancer cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1310-1

    TBL1 and ZEB1 interact and cooperate for regulation of the CDH1 and ZEB1 promoters. a Reporter assay showing repression of the CDH1 promoter by Flag-TBL1 and HA-ZEB1 in NMuMG cells. b Reporter assay showing activation of the ZEB1 promoter by Flag-TBL1 and HA-ZEB1 in HEK293T cells. c TBL1 and ZEB1 bind to the CDH1 and ZEB1 promoters. HMEC-RAS-ZEB1 cells transfected with control siRNAs (siControl) or siRNAs against TBL1 (siTBL1) were subjected to chromatin immunoprecipitation assays with the indicated antibodies. Fold enrichment indicate occupancies relative to siControl. a – c Values are the average of n = 6 data from three independent experiments ± SD. * P
    Figure Legend Snippet: TBL1 and ZEB1 interact and cooperate for regulation of the CDH1 and ZEB1 promoters. a Reporter assay showing repression of the CDH1 promoter by Flag-TBL1 and HA-ZEB1 in NMuMG cells. b Reporter assay showing activation of the ZEB1 promoter by Flag-TBL1 and HA-ZEB1 in HEK293T cells. c TBL1 and ZEB1 bind to the CDH1 and ZEB1 promoters. HMEC-RAS-ZEB1 cells transfected with control siRNAs (siControl) or siRNAs against TBL1 (siTBL1) were subjected to chromatin immunoprecipitation assays with the indicated antibodies. Fold enrichment indicate occupancies relative to siControl. a – c Values are the average of n = 6 data from three independent experiments ± SD. * P

    Techniques Used: Reporter Assay, Activation Assay, Transfection, Chromatin Immunoprecipitation

    7) Product Images from "CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis"

    Article Title: CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis

    Journal: Oncogene

    doi: 10.1038/s41388-018-0402-1

    CAB39L interacted with LKB1/STRAD leading to the activation of LKB1. a Co-immunoprecipitation (Co-IP) of CAB39L with anti-Flag and anti-CAB39L in AGS-CAB39L and MKN74 cells, respectively, identified LKB1 and STRAD as the binding partners of CAB39L. b Reciprocal Co-IP confirmed the protein interaction between LKB1 and CAB39L in both AGS and MKN74 cell lines. c CAB39L overexpression in AGS and MKN45 cells induced p-LKB1; while knockdown of CAB39L in MKN74 cells reduced LKB1 phosphorylation. d LKB1 (STK11) silencing abolished the effect of CAB39L overexpression on AMPKα phosphorylation activation. e , f LKB1 silencing abolished tumor suppressive effect of CAB39L in BGC832 cells, as determined by e cell viability and f colony formation assays, suggesting that the effect of CAB39L was dependent on LKB1. (* P
    Figure Legend Snippet: CAB39L interacted with LKB1/STRAD leading to the activation of LKB1. a Co-immunoprecipitation (Co-IP) of CAB39L with anti-Flag and anti-CAB39L in AGS-CAB39L and MKN74 cells, respectively, identified LKB1 and STRAD as the binding partners of CAB39L. b Reciprocal Co-IP confirmed the protein interaction between LKB1 and CAB39L in both AGS and MKN74 cell lines. c CAB39L overexpression in AGS and MKN45 cells induced p-LKB1; while knockdown of CAB39L in MKN74 cells reduced LKB1 phosphorylation. d LKB1 (STK11) silencing abolished the effect of CAB39L overexpression on AMPKα phosphorylation activation. e , f LKB1 silencing abolished tumor suppressive effect of CAB39L in BGC832 cells, as determined by e cell viability and f colony formation assays, suggesting that the effect of CAB39L was dependent on LKB1. (* P

    Techniques Used: Activation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Over Expression

    8) Product Images from "Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells"

    Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.05.073

    TPP1-S and TPP1-L Recruit Telomerase and Protect Chromosome Ends (A) HeLa-EM2-11ht cells expressing TPP1-S, TPP1-L M87A, or TPP1ΔNOB were analyzed for telomerase recruitment to telomeres by immunofluorescence (IF) and fluorescence in situ hybridization (FISH). “FLAG (TPP1)” indicates IF signal of the indicated TPP1 construct at telomeres (green). Telomerase RNA (“TR”) was detected by FISH with a fluorescently tagged DNA probe (red). The “merge” panel depicts the extent of telomerase recruitment to telomeres (yellow). (B) Quantitation of telomerase recruitment data of which (A) is representative. For each clone, > 100 telomere foci were scored, and the mean percentage (bar) and SD (error bar) of FLAG-TPP1 foci containing TR was plotted for triplicate measurements. Statistical significance was scored with a two-tailed Student’s t test. (C) Pull-down of transiently expressed FLAG-POT1 on anti-FLAG-conjugated beads with Myc-TPP1 and Myc-TIN2 constructs. (D) DNA binding curves from filter binding analysis of increasing concentrations of purified POT1 protein in the absence of any TPP1 protein or in the presence of 200 nM of either TPP1-S 90–334 or TPP1-L 1–334 . 10 pM 32 P-end-labeled GGTTAGG GTTAG DNA oligonucleotide was used in all binding experiments. .
    Figure Legend Snippet: TPP1-S and TPP1-L Recruit Telomerase and Protect Chromosome Ends (A) HeLa-EM2-11ht cells expressing TPP1-S, TPP1-L M87A, or TPP1ΔNOB were analyzed for telomerase recruitment to telomeres by immunofluorescence (IF) and fluorescence in situ hybridization (FISH). “FLAG (TPP1)” indicates IF signal of the indicated TPP1 construct at telomeres (green). Telomerase RNA (“TR”) was detected by FISH with a fluorescently tagged DNA probe (red). The “merge” panel depicts the extent of telomerase recruitment to telomeres (yellow). (B) Quantitation of telomerase recruitment data of which (A) is representative. For each clone, > 100 telomere foci were scored, and the mean percentage (bar) and SD (error bar) of FLAG-TPP1 foci containing TR was plotted for triplicate measurements. Statistical significance was scored with a two-tailed Student’s t test. (C) Pull-down of transiently expressed FLAG-POT1 on anti-FLAG-conjugated beads with Myc-TPP1 and Myc-TIN2 constructs. (D) DNA binding curves from filter binding analysis of increasing concentrations of purified POT1 protein in the absence of any TPP1 protein or in the presence of 200 nM of either TPP1-S 90–334 or TPP1-L 1–334 . 10 pM 32 P-end-labeled GGTTAGG GTTAG DNA oligonucleotide was used in all binding experiments. .

    Techniques Used: Expressing, Immunofluorescence, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Construct, Quantitation Assay, Two Tailed Test, Binding Assay, Purification, Labeling

    9) Product Images from "Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-β to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively *"

    Article Title: Kelch-like Protein 21 (KLHL21) Targets IκB Kinase-β to Regulate Nuclear Factor κ-Light Chain Enhancer of Activated B Cells (NF-κB) Signaling Negatively *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.715854

    KLHL21 protein specifically binds to IKKβ . A , 293T cells were transfected with KLHL21–3F. KLHL21-3F was immunoprecipitated with anti-FLAG beads and then analyzed by immunoblotting with the indicated antibodies. B , whole cell lysates prepared from 293T and RAW264.7 cells were immunoprecipitated with anti-KLHL21 polyclonal antibody and then analyzed by immunoblotting with the indicated antibodies. C , purified GST-KLHL21 fusion protein was incubated with whole cell lysates prepared from 293T and RAW264.7 cells, respectively. The precipitates after pull-down were analyzed by Western blotting with anti-KLHL21 antibody. D , HeLa cells were transfected with C-terminal mCherry-tagged KLHL21 ( red ). Cells were fixed and immunostained with anti-IKKβ antibody ( green ). E , 293T cells were cotransfected with vector expressing IKKα-3F, IKKβ-3F, NEMO-3F, and KLHL21-EE as indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblot with anti-EE and anti-FLAG, respectively. F , 293T cells cotransfected with IKKβ-EE and KLHL21–3F were collected at the indicated time points after TNFα treatment. KLHL21-3F was immunoprecipitated with anti-FLAG beads and then analyzed by immunoblotting with the indicated antibodies. The density ratio of immunoprecipitated IKKβ/KLHL21 quantified by densitometric scanning is shown on the right ( n = 3). All experiments were performed at least three times. IB , immunoblotting; IP , immunoprecipitation; WCL , whole cell lysates. Error bars , S.E.
    Figure Legend Snippet: KLHL21 protein specifically binds to IKKβ . A , 293T cells were transfected with KLHL21–3F. KLHL21-3F was immunoprecipitated with anti-FLAG beads and then analyzed by immunoblotting with the indicated antibodies. B , whole cell lysates prepared from 293T and RAW264.7 cells were immunoprecipitated with anti-KLHL21 polyclonal antibody and then analyzed by immunoblotting with the indicated antibodies. C , purified GST-KLHL21 fusion protein was incubated with whole cell lysates prepared from 293T and RAW264.7 cells, respectively. The precipitates after pull-down were analyzed by Western blotting with anti-KLHL21 antibody. D , HeLa cells were transfected with C-terminal mCherry-tagged KLHL21 ( red ). Cells were fixed and immunostained with anti-IKKβ antibody ( green ). E , 293T cells were cotransfected with vector expressing IKKα-3F, IKKβ-3F, NEMO-3F, and KLHL21-EE as indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblot with anti-EE and anti-FLAG, respectively. F , 293T cells cotransfected with IKKβ-EE and KLHL21–3F were collected at the indicated time points after TNFα treatment. KLHL21-3F was immunoprecipitated with anti-FLAG beads and then analyzed by immunoblotting with the indicated antibodies. The density ratio of immunoprecipitated IKKβ/KLHL21 quantified by densitometric scanning is shown on the right ( n = 3). All experiments were performed at least three times. IB , immunoblotting; IP , immunoprecipitation; WCL , whole cell lysates. Error bars , S.E.

    Techniques Used: Transfection, Immunoprecipitation, Purification, Incubation, Western Blot, Plasmid Preparation, Expressing

    KLHL21 directly binds to the kinase domain of IKKβ via its Kelch domains. A , 293T cells were cotransfected with KLHL21–3F, WT, and deletion mutants of IKKβ-EE, respectively, as indicated. KLHL21–3F was immunoprecipitated with anti-FLAG beads and blotted with anti-EE and anti-FLAG. ULD , ubiquitin-like domain; SDD , scaffold/dimerization domain; NBD , NEMO-binding domain. B , 293T cells were cotransfected with IKKβ-EE and the indicated KLHL21-3F deletion constructs. KLHL21-3F was immunoprecipitated with anti-FLAG beads and blotted with anti-EE and anti-FLAG. Results are representative of three independent experiments. C , 293T cells were transfected with the indicated KLHL21 deletion constructs. KLHL21-3F was immunoprecipitated with anti-FLAG beads and blotted with anti-Cul3 and anti-FLAG. D , 293T cells were cotransfected with EE-tagged wild-type IKKβ and IKKβ E39A mutant, along with FLAG-tagged KLHL21 and KEAP1 where indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblotting with anti-EE and anti-FLAG, respectively. IB , immunoblotting; IP , immunoprecipitation; WCL , whole cell lysates.
    Figure Legend Snippet: KLHL21 directly binds to the kinase domain of IKKβ via its Kelch domains. A , 293T cells were cotransfected with KLHL21–3F, WT, and deletion mutants of IKKβ-EE, respectively, as indicated. KLHL21–3F was immunoprecipitated with anti-FLAG beads and blotted with anti-EE and anti-FLAG. ULD , ubiquitin-like domain; SDD , scaffold/dimerization domain; NBD , NEMO-binding domain. B , 293T cells were cotransfected with IKKβ-EE and the indicated KLHL21-3F deletion constructs. KLHL21-3F was immunoprecipitated with anti-FLAG beads and blotted with anti-EE and anti-FLAG. Results are representative of three independent experiments. C , 293T cells were transfected with the indicated KLHL21 deletion constructs. KLHL21-3F was immunoprecipitated with anti-FLAG beads and blotted with anti-Cul3 and anti-FLAG. D , 293T cells were cotransfected with EE-tagged wild-type IKKβ and IKKβ E39A mutant, along with FLAG-tagged KLHL21 and KEAP1 where indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblotting with anti-EE and anti-FLAG, respectively. IB , immunoblotting; IP , immunoprecipitation; WCL , whole cell lysates.

    Techniques Used: Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis

    KLHL21 does not interfere with the interaction between IKKβ and TRAF2, TAK1, or IκBα . A , 293T cells were cotransfected with vector expressing IKKβ-EE, TBK1-EE, TRAF2-EE, TRAF6-EE, and KLHL21-3F as indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblotting with anti-EE and anti-FLAG, respectively. B–D , IKKβ and KLHL21 were coexpressed with TRAF2 ( B ), TAK1 ( C ), or undegradable IκBα mutant ( D ), respectively. The interactions between IKKβ and TRAF2, TAK1, or IκBα were analyzed by coimmunoprecipitation. WCL , whole cell lysates; IP , immunoprecipitation; IB , immunoblotting. Error bars , S.E.
    Figure Legend Snippet: KLHL21 does not interfere with the interaction between IKKβ and TRAF2, TAK1, or IκBα . A , 293T cells were cotransfected with vector expressing IKKβ-EE, TBK1-EE, TRAF2-EE, TRAF6-EE, and KLHL21-3F as indicated. Lysates were immunoprecipitated with anti-FLAG and then analyzed by immunoblotting with anti-EE and anti-FLAG, respectively. B–D , IKKβ and KLHL21 were coexpressed with TRAF2 ( B ), TAK1 ( C ), or undegradable IκBα mutant ( D ), respectively. The interactions between IKKβ and TRAF2, TAK1, or IκBα were analyzed by coimmunoprecipitation. WCL , whole cell lysates; IP , immunoprecipitation; IB , immunoblotting. Error bars , S.E.

    Techniques Used: Plasmid Preparation, Expressing, Immunoprecipitation, Mutagenesis

    10) Product Images from "Defective RNA polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 pathway"

    Article Title: Defective RNA polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 pathway

    Journal: eLife

    doi: 10.7554/eLife.35447

    Pol III is repressed by ubiquitylation and p97/Cdc48. ( A–C ) The indicated rpc160 mutant strains were crossed with slx5Δ , slx8Δ , or ubc4Δ strain, respectively, followed by tetrad analysis. The cross between slx8Δ and rpc160-G1297D was shown, because slx8Δ caused obvious growth defect by itself, so the rescue effect was more obvious on rpc160-G1297D, which is a sicker mutant than rpc160-M809I . ( D ) Yeast two-hybrid interactions between Slx5 and Rpc53. SLX5 and RPC53 were cloned into a 2μ LEU2 Gal4 activation-domain (AD) vector and a 2μ TRP1 DNA-binding domain (BD) vector, respectively, and co-transformed into yeast strain PJ69-4A. Transformants were selected on synthetic media lacking leucine and tryptophan (SC-LW), then patched and replica plated to selective media lacking histidine to test for interactions. The histidine-lacking media was supplement with 3-aminotriazole (SC-LWH + 3AT) for a more stringent phenotype. ( E ) LEU2 plasmids carrying HA-tagged wild type or SIM-defective SLX5 ( HA-slx5-sim ) were transformed into an rpc160-G1297D slx5Δ strain containing a URA3 RPC160 plasmid. Transformants were selected on SC-L then spotted onto an SC-L + 5 FOA plate to lose wild-type RPC160. HA-SLX5 complemented slx5Δ so the cells became sicker compared to the empty vector control transformants, while HA-slx5-sim did not complement, indicating that the SIMs are essential for the function of SLX5 in this assay. The lost Slx5 function by the SIM mutations was not caused by insufficient proteins, since there were comparable levels of Slx5 proteins, as determined by an anti-HA immunoblot on total cell lysates (right, top panel). G6PDH served as a loading control (right, bottom panel). ( F ) Tetrad analysis between rpc160-M809I and cdc48-3 . ( G ) Determination of Rpc160 association with other Pol III subunits. Pol III complexes containing Flag-tagged wild type or mutant Rpc160 in wild-type CDC48 or cdc48-3 cells were isolated using anti-Flag agarose beads, followed by TMS labeling and mass-spec analysis to quantify the relative amounts of Rpc160-interacting proteins. Signals for 12 of the 17 Pol III subunits, including Rpc160, were detected, and normalized to the signals from RPC160-Flag cells. n = Number of times when a unique peptide for the indicated protein is measured. An untagged RPC160 strain was used as a negative control. ( H ) ChIP analysis of Rpc160. Flag-tagged RPC160 or rpc160-M809I was expressed from a plasmid in wild-type CDC48 or cdc48-3 cells, as indicated. The Flag-tagged proteins were purified using anti-Flag agarose beads. An untagged strain was used as negative control. Three tRNA gene loci, as well as 18S rDNA (negative control), were examined. Chromatin association was determined by real-time PCR of the indicated genomic loci, using the percent of input method. Data are mean ± standard deviation calculated from six data points (two biological replicates and three technical replicates).
    Figure Legend Snippet: Pol III is repressed by ubiquitylation and p97/Cdc48. ( A–C ) The indicated rpc160 mutant strains were crossed with slx5Δ , slx8Δ , or ubc4Δ strain, respectively, followed by tetrad analysis. The cross between slx8Δ and rpc160-G1297D was shown, because slx8Δ caused obvious growth defect by itself, so the rescue effect was more obvious on rpc160-G1297D, which is a sicker mutant than rpc160-M809I . ( D ) Yeast two-hybrid interactions between Slx5 and Rpc53. SLX5 and RPC53 were cloned into a 2μ LEU2 Gal4 activation-domain (AD) vector and a 2μ TRP1 DNA-binding domain (BD) vector, respectively, and co-transformed into yeast strain PJ69-4A. Transformants were selected on synthetic media lacking leucine and tryptophan (SC-LW), then patched and replica plated to selective media lacking histidine to test for interactions. The histidine-lacking media was supplement with 3-aminotriazole (SC-LWH + 3AT) for a more stringent phenotype. ( E ) LEU2 plasmids carrying HA-tagged wild type or SIM-defective SLX5 ( HA-slx5-sim ) were transformed into an rpc160-G1297D slx5Δ strain containing a URA3 RPC160 plasmid. Transformants were selected on SC-L then spotted onto an SC-L + 5 FOA plate to lose wild-type RPC160. HA-SLX5 complemented slx5Δ so the cells became sicker compared to the empty vector control transformants, while HA-slx5-sim did not complement, indicating that the SIMs are essential for the function of SLX5 in this assay. The lost Slx5 function by the SIM mutations was not caused by insufficient proteins, since there were comparable levels of Slx5 proteins, as determined by an anti-HA immunoblot on total cell lysates (right, top panel). G6PDH served as a loading control (right, bottom panel). ( F ) Tetrad analysis between rpc160-M809I and cdc48-3 . ( G ) Determination of Rpc160 association with other Pol III subunits. Pol III complexes containing Flag-tagged wild type or mutant Rpc160 in wild-type CDC48 or cdc48-3 cells were isolated using anti-Flag agarose beads, followed by TMS labeling and mass-spec analysis to quantify the relative amounts of Rpc160-interacting proteins. Signals for 12 of the 17 Pol III subunits, including Rpc160, were detected, and normalized to the signals from RPC160-Flag cells. n = Number of times when a unique peptide for the indicated protein is measured. An untagged RPC160 strain was used as a negative control. ( H ) ChIP analysis of Rpc160. Flag-tagged RPC160 or rpc160-M809I was expressed from a plasmid in wild-type CDC48 or cdc48-3 cells, as indicated. The Flag-tagged proteins were purified using anti-Flag agarose beads. An untagged strain was used as negative control. Three tRNA gene loci, as well as 18S rDNA (negative control), were examined. Chromatin association was determined by real-time PCR of the indicated genomic loci, using the percent of input method. Data are mean ± standard deviation calculated from six data points (two biological replicates and three technical replicates).

    Techniques Used: Mutagenesis, Clone Assay, Activation Assay, Plasmid Preparation, Binding Assay, Transformation Assay, Isolation, Labeling, Mass Spectrometry, Negative Control, Chromatin Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Standard Deviation

    11) Product Images from "Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex"

    Article Title: Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.152611

    Interaction between Dnd1 and eIF3f is essential for nanos1 translation. (A) FLAG-tagged eIF3f deletions were transfected into HEK293T cells along with myc-Dnd1. Lysates were immunoprecipitated with anti-FLAG antibody and analyzed by western blot. The minimal Dnd1-binding domain was mapped to residues 92-200 of eIF3f, which is indicated by the red asterisk. (B) Schematic summarizing experiments shown in A. +, Dnd1 binding; −, lack of Dnd1 binding. (C) Myc-Dnd1 and FLAG-eIF3f were transfected into HEK293T cells with increasing amounts of eIF3f 92-200 . Cell lysates were immunoprecipitated with an anti-myc antibody and analyzed by western blot. (D) Myc-Dnd1 and FLAG-eIF3h were transfected into HEK293T cells in the presence or absence of eIF3f 92-200 . Cell lysates were immunoprecipitated with an anti-myc antibody and analyzed by western blot. Experiments were repeated three times. (E) nanos1 RNA was injected into fertilized eggs alone or with eIF3f 92-200 , or with eIF3f 92-200 and myc-dnd1 RNAs. At stage 11, Nanos1 protein was immunoprecipitated and analyzed by western blot. Non-specific band served as a loading control. The experiment was performed twice.
    Figure Legend Snippet: Interaction between Dnd1 and eIF3f is essential for nanos1 translation. (A) FLAG-tagged eIF3f deletions were transfected into HEK293T cells along with myc-Dnd1. Lysates were immunoprecipitated with anti-FLAG antibody and analyzed by western blot. The minimal Dnd1-binding domain was mapped to residues 92-200 of eIF3f, which is indicated by the red asterisk. (B) Schematic summarizing experiments shown in A. +, Dnd1 binding; −, lack of Dnd1 binding. (C) Myc-Dnd1 and FLAG-eIF3f were transfected into HEK293T cells with increasing amounts of eIF3f 92-200 . Cell lysates were immunoprecipitated with an anti-myc antibody and analyzed by western blot. (D) Myc-Dnd1 and FLAG-eIF3h were transfected into HEK293T cells in the presence or absence of eIF3f 92-200 . Cell lysates were immunoprecipitated with an anti-myc antibody and analyzed by western blot. Experiments were repeated three times. (E) nanos1 RNA was injected into fertilized eggs alone or with eIF3f 92-200 , or with eIF3f 92-200 and myc-dnd1 RNAs. At stage 11, Nanos1 protein was immunoprecipitated and analyzed by western blot. Non-specific band served as a loading control. The experiment was performed twice.

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

    Dnd1 physically interacts with eIF3f. (A) Anti-FLAG (lanes 4-6) or anti-Myc (lanes 10-12) antibodies were used to IP eIF3f or Dnd1 from cell lysates (lanes 9,12). Addition of RNaseA to lysates did not disrupt the Dnd1-eIF3f complex (lane 12). (B) Co-IP shows the interaction between myc-Dnd1 and endogenous eIF3f in HEK293T cells. IgG served as negative control. (C) Co-IP shows that myc-Dnd1 formed complexes with FLAG-eIF3 h and FLAG-eIF3m in HEK293T cells. Experiments were repeated three times.
    Figure Legend Snippet: Dnd1 physically interacts with eIF3f. (A) Anti-FLAG (lanes 4-6) or anti-Myc (lanes 10-12) antibodies were used to IP eIF3f or Dnd1 from cell lysates (lanes 9,12). Addition of RNaseA to lysates did not disrupt the Dnd1-eIF3f complex (lane 12). (B) Co-IP shows the interaction between myc-Dnd1 and endogenous eIF3f in HEK293T cells. IgG served as negative control. (C) Co-IP shows that myc-Dnd1 formed complexes with FLAG-eIF3 h and FLAG-eIF3m in HEK293T cells. Experiments were repeated three times.

    Techniques Used: Co-Immunoprecipitation Assay, Negative Control

    12) Product Images from "PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair"

    Article Title: PAXX and its paralogs synergistically direct DNA polymerase λ activity in DNA repair

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06127-y

    Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG-Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed
    Figure Legend Snippet: Pol λ interacts with XRCC4 family proteins via its BRCT domain in cells. a HEK293F cells were irradiated with 10 Gy X-ray or left untreated. Soluble nuclear extracts were isolated following 0–60 min post-irradiation recovery time at 37 °C. Following IP with anti-Pol λ or rabbit IgG (rIgG), Pol λ and associated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. b HEK293F cell nucleoplasmic (NP) or soluble chromatin (sol. Chr) extracts were immunoprecipitated with rIgG, anti-PAXX or -XLF or mouse IgG (mIgG) or anti-XRCC4. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted for the indicated NHEJ factors. c As described in Panel A, except that soluble nuclear extracts were incubated with 0-200 μg/ml EtBr for 1 h prior to IP with anti-Pol λ or rIgG. d EMSA showing that interaction of Pol λ with DNA-bound Ku requires R57 and L60 in the BRCT domain of Pol λ. Reactions were performed with IRDye® 700-labelled 5nt-gapped dsDNA (33-mer) in the presence or absence of FLAG-Ku70/80 (20 nM) and either FLAG-Pol λ-WT or a R57A/L60A mutant (50 nM). e HEK293F cells were transiently transfected with either pCMX-LacZ (control) or pCMX-FLAG-Pol λ-WT, -ΔBRCT or a R57A/L60A mutant and anti-FLAG IPs performed

    Techniques Used: Irradiation, Isolation, SDS Page, Non-Homologous End Joining, Immunoprecipitation, Incubation, Mutagenesis, Transfection

    13) Product Images from "Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2"

    Article Title: Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06160-x

    Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies
    Figure Legend Snippet: Eukaryotic-specific segments of eS26 are required to bind Tsr2. a XL-MS reveals crosslinks between ESS2 and N-terminal domain of Tsr2. The crosslinked residues are listed in the Supplementary Table 1 . b Phylogenetic analyses for eS26 and Tsr2. ESS1, ESS2 from eS26 and Tsr2 are present only in eukaryotes. c Sequence alignment of yeast S26 compared to the indicated species. 70 d ESSs in eS26 are required to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant WT eS26, eS26 deficient in ESS1 and/or ESS2 or archaeal eS26 from Sulfolobus solfataricus . Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE and visualized by Coomassie Blue staining. L = input (1:10 diluted). e Residues 99–109 in eS26-ESS2 are necessary to bind Tsr2 in vitro. GST-Tsr2 was immobilized on Glutathione Sepharose before incubation with an E. coli lysate containing recombinant WT eS26 or eS26 with variant truncations in C-terminal ESS2. Samples were analyzed as in d . Results from in vitro binding were quantified using ImageJ. f ESS1 and ESS2 deletion from eS26 causes slow growth phenotype in yeast. The conditional P GAL1 - RPS26Arps26b∆ strain was transformed with WT or the indicated truncations of eS26 and spotted in 10-fold dilutions on repressive glucose containing media and grown at 25 °C for 4 days. g Cells with eS26 lacking ESS1 or ESS2 accumulate immature 20S pre-rRNA in the cytoplasm. Localization of 20S pre-rRNA in P GAL1 - RPS26Arps26b∆ cells transformed with indicated plasmids was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. h Overexpression of ProtA-FLAG-eS26 is toxic in yeast. The WT yeast strain (BY4741) was transformed with ProtA-FLAG-eS26 or ProtA-FLAG-eS26 lacking ESS2, spotted in 10-fold dilutions on galactose containing media and grown at 25 °C for 4 days. i FLAG-ESS2 fusion protein co-precipitates Tsr2. ESS2 was purified using ProteinA-Tev-FLAG tag, the FLAG eluate was TCA precipitated, separated by SDS-PAGE, and analyzed by Coomassie staining and western analyses using the indicated antibodies

    Techniques Used: Mass Spectrometry, Sequencing, In Vitro, Incubation, Recombinant, SDS Page, Staining, Variant Assay, Binding Assay, Transformation Assay, Fluorescence In Situ Hybridization, Labeling, Over Expression, Purification, FLAG-tag, Western Blot

    ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control
    Figure Legend Snippet: ESSs mediate Tsr2-dependent importin:eS26 complex disassembly. a Tsr2 cannot efficiently dissociate the Kap123:eS26ΔESS1ΔESS2 FLAG complex. The complex of GST-Kap123 with eS26 FLAG , eS26ΔESS1 FLAG eS26ΔESS2 FLAG or eS26ΔESS1ΔESS2 FLAG was immobilized on Glutathione Sepharose and incubated with either buffer alone or with Tsr2 before pull-down. L = input (1:10 diluted). b Tsr2 DWI mutant does not dissociate a Kap123:eS26 complex. The complex of GST-Kap123 with eS26 FLAG was incubated with either buffer alone, purified Tsr2 or Tsr2 DWI before pull-down. L = input (1:10 diluted). c Efficient recruitment of eS26 to pre-40S requires both of the ESSs. Enp1-TAP was isolated from P GAL1 - RPS26Arps26b∆ strain transformed with WT eS26 or eS26 lacking ESS1 or ESS2. After tandem affinity purification, eluates were separated by 4–12% gradient SDS-PAGE and subjected to western analyses using indicated antibodies. Protein levels of uS7, uS3 served as a loading control. d Protein levels of eS26, eS26ΔESS1, eS26ΔESS2 and eS26ΔESS1ΔESS2 in whole-cell extracts of P GAL1 - RPS26Arps26b∆ strain were determined by western analyses using α-eS26 antibodies. Gsp1 protein levels served as a loading control

    Techniques Used: Incubation, Mutagenesis, Purification, Isolation, Transformation Assay, Affinity Purification, SDS Page, Western Blot

    14) Product Images from "Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export"

    Article Title: Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export

    Journal: Genes & Development

    doi: 10.1101/gad.328690.119

    CG13741 and Nxf3 impact dual-strand piRNA cluster biology. ( A ) Schematic representation of CG13741 as well as CG13741 Δ1 and CG13741 Δ2 mutants. ( B ) Cartoon displaying the Nxf3 domain structure and the generated nxf3 mut allele. Peptide coverage obtained by Nxf3 pull-down followed by mass spectrometry is indicated in blue. ( C ) Bar graphs showing fold changes in steady-state RNA levels of a housekeeping gene ( act5c ) and soma-specific ( gypsy ), intermediate ( blood ), and germline-specific ( HeT-A and burdock ) transposons in total ovarian RNA from the indicated genotypes (relative to heterozygote siblings and normalized to rp49 ). Error bars indicate standard deviation. n = 3. ( D ) Expression and localization of CG13741 in an egg chamber of control and CG13741 mut mutant flies are shown by immunofluorescence. A zoomed-in view of the indicated nurse cell nucleus is shown at the right . (Green) CG13741; (magenta) Lamin; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm. ( E ) As in D but showing the expression and localization of Nxf3 in control and nxf3 mut egg chambers. (Green) Nxf3; (magenta) Lamin; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm. ( F ) As in D but showing expression and localization of GFP-tagged CG13741 and GFP-tagged Nxf3 costained with Rhi. (Green) GFP; (red) Rhi; (blue) DNA. Scale bar, 2 µm. ( G ) As in D but showing expression and localization of the annotated and extended Nxf3 sequence tagged C-terminally with GFP-3xFlag and costained with an antibody to endogenous Nxf3. (Green) Nxf3; (red) Flag; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm.
    Figure Legend Snippet: CG13741 and Nxf3 impact dual-strand piRNA cluster biology. ( A ) Schematic representation of CG13741 as well as CG13741 Δ1 and CG13741 Δ2 mutants. ( B ) Cartoon displaying the Nxf3 domain structure and the generated nxf3 mut allele. Peptide coverage obtained by Nxf3 pull-down followed by mass spectrometry is indicated in blue. ( C ) Bar graphs showing fold changes in steady-state RNA levels of a housekeeping gene ( act5c ) and soma-specific ( gypsy ), intermediate ( blood ), and germline-specific ( HeT-A and burdock ) transposons in total ovarian RNA from the indicated genotypes (relative to heterozygote siblings and normalized to rp49 ). Error bars indicate standard deviation. n = 3. ( D ) Expression and localization of CG13741 in an egg chamber of control and CG13741 mut mutant flies are shown by immunofluorescence. A zoomed-in view of the indicated nurse cell nucleus is shown at the right . (Green) CG13741; (magenta) Lamin; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm. ( E ) As in D but showing the expression and localization of Nxf3 in control and nxf3 mut egg chambers. (Green) Nxf3; (magenta) Lamin; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm. ( F ) As in D but showing expression and localization of GFP-tagged CG13741 and GFP-tagged Nxf3 costained with Rhi. (Green) GFP; (red) Rhi; (blue) DNA. Scale bar, 2 µm. ( G ) As in D but showing expression and localization of the annotated and extended Nxf3 sequence tagged C-terminally with GFP-3xFlag and costained with an antibody to endogenous Nxf3. (Green) Nxf3; (red) Flag; (blue) DNA. Scale bar for egg chamber, 10 µm; scale bar for zoom-in, 2 µm.

    Techniques Used: Generated, Mass Spectrometry, Standard Deviation, Expressing, Mutagenesis, Immunofluorescence, Sequencing

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    Autoradiography:

    Article Title: The intracellular NADH level regulates atrophic nonunion pathogenesis through the CtBP2-p300-Runx2 transcriptional complex
    Article Snippet: .. The blots were probed with primary anti-Flag (Sigma, USA), anti-Myc (Sigma, #F1804), anti-CtBP2 (BD Biosciences, USA, #612044), anti-p300 (Santa Cruz Biotechnology, USA, #sc-585), anti-GAPDH (Santa Cruz Biotechnology, #sc-365062), or anti-Runx2 (Santa Cruz Biotechnology, #sc-101145) antibodies, and the chemiluminescence signals were detected by autoradiography. .. The total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, #15596026) following the manufacturer's instructions.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2
    Article Snippet: Antibodies For immunoprecipitation the following antibodies were used: mouse Flag M2 (Sigma, Cat# F1804, 1 μg/mg total proteins). .. For immunoblotting analysis the following antibodies were used: Flag M2, HRP conjugated (Sigma, Cat# F1804, 1:10,000), rabbit pan-14-3-3 Santa Cruz, Cat# sc-629, 1:1,000), mouse monoclonal Anti-polyHistidine, HRP conjugated (Sigma, CatA7058, 1:10,000), rabbit phospho-PAK4-5-6 (Sigma, Cat# SAB4504052, 1:2,000), mouse β-tubulin (Sigma, Cat# T8328, 1:5,000), rabbit pan phospho-Ser58 14-3-3 (Abcam, Cat# ab30554, 1:1,000), rabbit 14–3–3γ (ThermoFisher, Cat# PA5-29690, 1:10,000), rabbit MJFF2 (Epitomics Cat# 3514-1, 1:100), rabbit phospho-Ser935 (Epitomics Cat# 5099-1, 1:100), mouse GFP (Roche, Cat# 1181460001, 1:1,000). .. For immunocytochemistry analysis the following antibodies were used: rabbit PAK6 (Prestige® Sigma, Cat# HPA031124, 1:200), rabbit 14–3–3γ (ThermoFisher, Cat# PA5-29690, 1:200), rabbit pan phospho-Ser58 14-3-3 (Abcam, Cat# ab30554, 1:200).

    FLAG-tag:

    Article Title: GILT restricts the cellular entry mediated by the envelope glycoproteins of SARS-CoV, Ebola virus and Lassa fever virus
    Article Snippet: Human Interferon alpha-2b (IFN-α2b) and IFN-γ were ordered from PBL Interferon Source (Catalog No. 11105–1 and 11500-1, respectively). .. Monoclonal antibody against FLAG tag (ANTI-FLAG M2) and β-Actin were purchased from Sigma (Catalog No. F1804 and A2228, respectively). .. Rabbit polyclonal antibody against GILT was obtained from Sigma (HPA026650).

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    Millipore monoclonal anti flag m2 antibody
    Construction of an AAV9-based plasmid expressing the functional NPC1 protein. A: Diagram of the AAV9 vector expressing NPC1-3×Flag. B: Expression of exogenous NPC1 attenuates lysosomal cholesterol accumulation in CT43 cells. CT43 cells were transfected with AAV9-NPC1-3×Flag. Cholesterol and NPC1 protein were visualized by filipin staining (pseudocolored red) and the <t>anti-Flag</t> antibody (pseudocolored green), respectively. Nuclei were stained with SYBGreen (pseudocolored blue). Scale bar, 20 μm.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
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    monoclonal anti flag m2 antibody - by Bioz Stars, 2021-04
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    Construction of an AAV9-based plasmid expressing the functional NPC1 protein. A: Diagram of the AAV9 vector expressing NPC1-3×Flag. B: Expression of exogenous NPC1 attenuates lysosomal cholesterol accumulation in CT43 cells. CT43 cells were transfected with AAV9-NPC1-3×Flag. Cholesterol and NPC1 protein were visualized by filipin staining (pseudocolored red) and the anti-Flag antibody (pseudocolored green), respectively. Nuclei were stained with SYBGreen (pseudocolored blue). Scale bar, 20 μm.

    Journal: Journal of Lipid Research

    Article Title: AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease [S]

    doi: 10.1194/jlr.M071274

    Figure Lengend Snippet: Construction of an AAV9-based plasmid expressing the functional NPC1 protein. A: Diagram of the AAV9 vector expressing NPC1-3×Flag. B: Expression of exogenous NPC1 attenuates lysosomal cholesterol accumulation in CT43 cells. CT43 cells were transfected with AAV9-NPC1-3×Flag. Cholesterol and NPC1 protein were visualized by filipin staining (pseudocolored red) and the anti-Flag antibody (pseudocolored green), respectively. Nuclei were stained with SYBGreen (pseudocolored blue). Scale bar, 20 μm.

    Article Snippet: The primary antibodies used were as follows: rabbit polyclonal anti-calnexin (catalog number 2433; Cell Signaling Technology, Danvers, MA); mouse monoclonal anti-clathrin heavy chain (catalog number 610499; BD Transduction Laboratories, San Jose, CA); mouse monoclonal anti-calbindin-D28K (catalog number C9848; Sigma); mouse monoclonal anti-Flag (catalog number F1804; Sigma); Alexa Fluor 555 goat anti-mouse IgG (1:500; catalog number ) and Alexa Fluor 488 goat anti-mouse IgG (1:500; catalog number A11001) were purchased from Life Technologies (Carlsbad, CA); horseradish peroxidase-conjugated goat anti-mouse (1:5,000; catalog number 31430) and anti-rabbit (1:5,000; catalog number 32460) IgG were purchased from Thermo Fisher Scientific (Waltham, MA).

    Techniques: Plasmid Preparation, Expressing, Functional Assay, Transfection, Staining