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    Structured Review

    Millipore immunoprecipitation buffer
    Nucleosome assembly is required for fork progression. Artificial blocking of Asf1–CAF-1–mediated nucleosome assembly by transient expression of the HIRA-B domain fused to an SV40 NLS. (A) <t>Immunoprecipitation</t> (IP) of FLAG-HA–tagged HIRA-B and HIRA-B mt carrying three point mutations disabling Asf1 binding. (B) Cell cycle profiles. GFP-spectrin was used to identify transfected cells by FACS. One representative experiment out of five biological replicas is shown. (C and D) EdU incorporation (C) and chromatin-bound RPA (D) detected by immunofluorescence in preextracted cells. Cells were cotransfected with H2B-GFP to identify transfected cells and treated 1 h with HU where indicated. Error bars indicate SDs of four biological replicas. Bars, 20 µm. (E) Single-cell analysis of DNA replication and DNA damage in U-2-OS cells transfected with full-length HIRA or the HIRA-B domain for 24 h. A dot plot of EdU and γ-H2AX intensities (left) and a scatter plot of γ-H2AX intensities (right) are shown. Cells treated 2 h with HU were included as a positive control. Cells were pulsed 15 min with EdU. Lines represent medians. n > 70. ***, P
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    Images

    1) Product Images from "New histone supply regulates replication fork speed and PCNA unloading"

    Article Title: New histone supply regulates replication fork speed and PCNA unloading

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305017

    Nucleosome assembly is required for fork progression. Artificial blocking of Asf1–CAF-1–mediated nucleosome assembly by transient expression of the HIRA-B domain fused to an SV40 NLS. (A) Immunoprecipitation (IP) of FLAG-HA–tagged HIRA-B and HIRA-B mt carrying three point mutations disabling Asf1 binding. (B) Cell cycle profiles. GFP-spectrin was used to identify transfected cells by FACS. One representative experiment out of five biological replicas is shown. (C and D) EdU incorporation (C) and chromatin-bound RPA (D) detected by immunofluorescence in preextracted cells. Cells were cotransfected with H2B-GFP to identify transfected cells and treated 1 h with HU where indicated. Error bars indicate SDs of four biological replicas. Bars, 20 µm. (E) Single-cell analysis of DNA replication and DNA damage in U-2-OS cells transfected with full-length HIRA or the HIRA-B domain for 24 h. A dot plot of EdU and γ-H2AX intensities (left) and a scatter plot of γ-H2AX intensities (right) are shown. Cells treated 2 h with HU were included as a positive control. Cells were pulsed 15 min with EdU. Lines represent medians. n > 70. ***, P
    Figure Legend Snippet: Nucleosome assembly is required for fork progression. Artificial blocking of Asf1–CAF-1–mediated nucleosome assembly by transient expression of the HIRA-B domain fused to an SV40 NLS. (A) Immunoprecipitation (IP) of FLAG-HA–tagged HIRA-B and HIRA-B mt carrying three point mutations disabling Asf1 binding. (B) Cell cycle profiles. GFP-spectrin was used to identify transfected cells by FACS. One representative experiment out of five biological replicas is shown. (C and D) EdU incorporation (C) and chromatin-bound RPA (D) detected by immunofluorescence in preextracted cells. Cells were cotransfected with H2B-GFP to identify transfected cells and treated 1 h with HU where indicated. Error bars indicate SDs of four biological replicas. Bars, 20 µm. (E) Single-cell analysis of DNA replication and DNA damage in U-2-OS cells transfected with full-length HIRA or the HIRA-B domain for 24 h. A dot plot of EdU and γ-H2AX intensities (left) and a scatter plot of γ-H2AX intensities (right) are shown. Cells treated 2 h with HU were included as a positive control. Cells were pulsed 15 min with EdU. Lines represent medians. n > 70. ***, P

    Techniques Used: Blocking Assay, Expressing, Immunoprecipitation, Binding Assay, Transfection, FACS, Recombinase Polymerase Amplification, Immunofluorescence, Single-cell Analysis, Positive Control

    2) Product Images from "Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress"

    Article Title: Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress

    Journal: Scientific Reports

    doi: 10.1038/srep16529

    Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p
    Figure Legend Snippet: Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

    Techniques Used: Immunoprecipitation, Western Blot

    3) Product Images from "Nucleosome stability mediated by histone variants H3.3 and H2A.Z"

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    Journal: Genes & Development

    doi: 10.1101/gad.1547707

    ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.
    Figure Legend Snippet: ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Immunoprecipitation

    4) Product Images from "Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway"

    Article Title: Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007882

    IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).
    Figure Legend Snippet: IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).

    Techniques Used: Transfection, Immunoprecipitation, Negative Control, In Vitro, Binding Assay, Incubation

    IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.
    Figure Legend Snippet: IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.

    Techniques Used: Transfection, Infection, Immunoprecipitation, Binding Assay, Negative Control, Staining

    IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.
    Figure Legend Snippet: IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.

    Techniques Used: In Vitro, Binding Assay, Negative Control, Incubation, SDS Page, Infection, Immunoprecipitation

    5) Product Images from "Critical Role of S1PR1 and Integrin ?4 in HGF/c-Met-mediated Increases in Vascular Integrity"

    Article Title: Critical Role of S1PR1 and Integrin ?4 in HGF/c-Met-mediated Increases in Vascular Integrity

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.404780

    c-Met/S1PR1/ITGB4 complex modulation by HGF and S1P. A , HPAEC were treated with either S1P (1 μ m ) or HGF (25 ng/ml) for 1 and 5 min. Cell lysates were subjected to immunoprecipitation with an anti-ITGB4 antibody and tyrosine or threonine phosphorylation
    Figure Legend Snippet: c-Met/S1PR1/ITGB4 complex modulation by HGF and S1P. A , HPAEC were treated with either S1P (1 μ m ) or HGF (25 ng/ml) for 1 and 5 min. Cell lysates were subjected to immunoprecipitation with an anti-ITGB4 antibody and tyrosine or threonine phosphorylation

    Techniques Used: Immunoprecipitation

    6) Product Images from "Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming"

    Article Title: Enhancer Analysis Unveils Genetic Interactions between TLX and SOX2 in Neural Stem Cells and In Vivo Reprogramming

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2015.09.015

    Transcription Factors Regulating Tlx Enhancer Activity (A) A diagram showing locations of the consensus transcription factor binding sequences (BS). (B) Diminished enhancer activity with mutations in the SOX2- or MYT1-binding sequences. Constitutively expressed tdTomato was used as an internal control for electroporation. The ratios of GFP + cells over tdTomato + cells are indicated in the parentheses (n = 3 mice; mean ± SEM). The scale bar represents 50 μm. (C) MYT1 directly binds to the identified enhancer. Antibody-induced supershift in electrophoresis mobility shift assays (EMSA) is shown in the boxed region. Normal IgG was used as controls for EMSA and chromatin immunoprecipitation (ChIP) assays (mean ± SEM; n = 3 independent experiments for control IgG and MYT1 antibody). (D) SOX2 directly binds to the identified enhancer. The boxed region shows antibody-induced supershift of the probe. Normal IgG was used as controls for EMSA and ChIP (mean ± SEM; n = 3 independent experiments for control IgG and SOX2 antibody). See also Table S2 .
    Figure Legend Snippet: Transcription Factors Regulating Tlx Enhancer Activity (A) A diagram showing locations of the consensus transcription factor binding sequences (BS). (B) Diminished enhancer activity with mutations in the SOX2- or MYT1-binding sequences. Constitutively expressed tdTomato was used as an internal control for electroporation. The ratios of GFP + cells over tdTomato + cells are indicated in the parentheses (n = 3 mice; mean ± SEM). The scale bar represents 50 μm. (C) MYT1 directly binds to the identified enhancer. Antibody-induced supershift in electrophoresis mobility shift assays (EMSA) is shown in the boxed region. Normal IgG was used as controls for EMSA and chromatin immunoprecipitation (ChIP) assays (mean ± SEM; n = 3 independent experiments for control IgG and MYT1 antibody). (D) SOX2 directly binds to the identified enhancer. The boxed region shows antibody-induced supershift of the probe. Normal IgG was used as controls for EMSA and ChIP (mean ± SEM; n = 3 independent experiments for control IgG and SOX2 antibody). See also Table S2 .

    Techniques Used: Activity Assay, Binding Assay, Electroporation, Mouse Assay, Electrophoresis, Mobility Shift, Chromatin Immunoprecipitation

    7) Product Images from "Regulation of lipid synthesis by the RNA helicase Mov10 controls Wnt5a production"

    Article Title: Regulation of lipid synthesis by the RNA helicase Mov10 controls Wnt5a production

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2015.15

    Mov10 directly regulates mRNA levels post transcriptionally. ( a–c ) FASN, SCD and Wnt5a levels in UACC903 cells expressing Mov10 shRNA (gray bars) or control shRNA (black bars) after treatment with actinomycin D (5 μg/ml) for 8 h. ( d–f ) Wnt5a, SCD and FASN mRNA detected in anti-Mov10 immunoprecipitation in UACC903 cells compared with control IgG.
    Figure Legend Snippet: Mov10 directly regulates mRNA levels post transcriptionally. ( a–c ) FASN, SCD and Wnt5a levels in UACC903 cells expressing Mov10 shRNA (gray bars) or control shRNA (black bars) after treatment with actinomycin D (5 μg/ml) for 8 h. ( d–f ) Wnt5a, SCD and FASN mRNA detected in anti-Mov10 immunoprecipitation in UACC903 cells compared with control IgG.

    Techniques Used: Expressing, shRNA, Immunoprecipitation

    8) Product Images from "Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *"

    Article Title: Dissecting the Molecular Pathway Involved in PLK2 Kinase-mediated α-Synuclein-selective Autophagic Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.759373

    α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p
    Figure Legend Snippet: α-Syn N-terminal region was required for its PLK2-mediated phosphorylation and degradation. A , Western blot illustrating the expression levels of α-syn and PLK2 in the presence of SPAR, which disrupts the formation of α-syn/PLK2 protein complex. B , quantification of α-syn protein levels showing that SPAR overexpression suppresses PLK2-mediated α-syn elimination ( n = 3). C , quantification of PLK2 protein levels showing that SPAR overexpression induced a significant accumulation of PLK2 ( n = 3). D , Western blot analysis of the total α-syn and Ser(P)-129 protein levels 24 h post transfection in HEK-239T cells. Cells were transiently transfected with 0.5 μg of α-syn Δ2–11 or 1 μg of α-syn Δ2–60 and 0.5 μg of PLK2 plasmids, and the total protein fraction was collected directly in 1X Laemmli buffer. E , histograms representing the quantification of α-syn protein levels, normalized against the actin expression, and showing that the N-terminal truncation Δ2–60, but not Δ2–11, affects α-syn degradation ( n = 3). F , histograms representing the quantification of Ser(P)-129 levels after PLK2 overexpression, normalized against total α-syn protein expression ( n = 3). The results show that Δ2–60 truncation induced a significant reduction of α-syn phosphorylation levels compared with α-syn Δ2–11. G , co-immunoprecipitation ( IP ) of PLK2 with N-terminal truncated α-syn (Δ2–11 or Δ2–60) showing that the deletion of the entire N-terminal regions (Δ2–60) is sufficient to block PLK2 and α-syn protein-protein transfection. *, p

    Techniques Used: Western Blot, Expressing, Over Expression, Transfection, Immunoprecipitation, Blocking Assay

    9) Product Images from "Immortalization of Mouse Germ Line Stem Cells"

    Article Title: Immortalization of Mouse Germ Line Stem Cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1634/stemcells.2003-0036

    Immunoprecipitation of GFRα-1, Dazl, and Oct- 4 using C18-4 protein extracts. (A): Immunoprecipitation of GFRα-1. Lane A: molecular weight markers; lane B: adult kidney; lane C: developing brain (6-day-old); lane D: adult testis; lane
    Figure Legend Snippet: Immunoprecipitation of GFRα-1, Dazl, and Oct- 4 using C18-4 protein extracts. (A): Immunoprecipitation of GFRα-1. Lane A: molecular weight markers; lane B: adult kidney; lane C: developing brain (6-day-old); lane D: adult testis; lane

    Techniques Used: Immunoprecipitation, Molecular Weight

    10) Product Images from "m6A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner"

    Article Title: m6A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1417-4

    Silencing of METTL3 elevates SOCS3 mRNA stability via YTHDF2-dependent mechanism. a Western blot analysis of FLAG, YTHDF2, and SOCS3 in piPSCs transfected with control and YTHDF2-FLAG plasmid. β-Actin was used as loading control. b RNA immunoprecipitation (RIP) analysis of the interaction of SOCS3 with FLAG in piPSCs transfected with YTHDF2-FLAG plasmid. Enrichment of SOCS3 with FLAG was measured by qPCR and normalized to input. c Relative luciferase activity of WT or MUT SOCS3-3′UTR luciferase reporter in piPSCs transfected with control or YTHDF2 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. d qPCR analysis of YTHDF2 in control and YTHDF2 knockdown piPSCs. GAPDH was used as an internal control. e Western blot analysis of SOCS3 and YTHDF2 in piPSCs with or without YTHDF2 knockdown. f mRNA stability analysis of SOCS3 mRNA in control, METTL3-depleted or YTHDF2-depleted piPSCs treated with actinomycin D for 3 and 6 h. g Western blot analysis of SOCS3, KLF4, and SOX2 in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. h AP staining of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. i Quantification of AP-positive colonies of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. j qPCR analysis of SOX2 and KLF4 expression in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. Data were presented as mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: Silencing of METTL3 elevates SOCS3 mRNA stability via YTHDF2-dependent mechanism. a Western blot analysis of FLAG, YTHDF2, and SOCS3 in piPSCs transfected with control and YTHDF2-FLAG plasmid. β-Actin was used as loading control. b RNA immunoprecipitation (RIP) analysis of the interaction of SOCS3 with FLAG in piPSCs transfected with YTHDF2-FLAG plasmid. Enrichment of SOCS3 with FLAG was measured by qPCR and normalized to input. c Relative luciferase activity of WT or MUT SOCS3-3′UTR luciferase reporter in piPSCs transfected with control or YTHDF2 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. d qPCR analysis of YTHDF2 in control and YTHDF2 knockdown piPSCs. GAPDH was used as an internal control. e Western blot analysis of SOCS3 and YTHDF2 in piPSCs with or without YTHDF2 knockdown. f mRNA stability analysis of SOCS3 mRNA in control, METTL3-depleted or YTHDF2-depleted piPSCs treated with actinomycin D for 3 and 6 h. g Western blot analysis of SOCS3, KLF4, and SOX2 in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. h AP staining of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. i Quantification of AP-positive colonies of piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. j qPCR analysis of SOX2 and KLF4 expression in piPSCs with or without METTL3 knockdown and transfected with control or YTHDF2 plasmid. Data were presented as mean ± SD of three independent experiments. ** P

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Staining, Expressing

    METTL3 regulates JAK2 and SOCS3 expression via m 6 A methylation. a m 6 A dot blot analysis in piPSCs transfected with control, wild-type (WT), and mutant (MUT) METTL3 plasmid. Equal loading of mRNA was verified by methylene blue staining (lower panel). b Western blot analysis of METTL3, JAK2, and SOCS3 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. β-Actin was used as loading control. c AP staining of piPSCs transfected with control, WT, and MUT METTL3 plasmid. d Quantification of AP-positive colonies of piPSCs transfected with control, WT, and MUT METTL3 plasmid. e qPCR analysis of piPSCs transfected with control, WT, and MUT METTL3 plasmid. GAPDH was used as an internal control. f Western blot analysis of KLF4 and SOX2 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. g Top consensus motif identified by HOMER with m 6 A-seq peaks in piPSCs. h Distribution of m 6 A peaks across the length of mRNA transcripts. Each region of 5′UTRs, CDSs, and 3′UTRs were binned into 100 segments, and the percentage of m 6 A peaks that fall within each bin was determined. i The m 6 A abundances in JAK2 and SOCS3 mRNA transcripts in piPSCs as detected by m 6 A-seq. The m 6 A peaks were shown in the black rectangles. j Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs with or without METTL3 knockdown. k MeRIP-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs transfected with control or METTL3 plasmid. l Relative luciferase activity of WT or MUT (A-to-T mutation) SOCS3-3′UTR (or JAK2-3′UTR) luciferase reporter in piPSCs transfected with control, WT, or MUT METTL3 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data were presented as mean ± SD of three independent experiments. ** P
    Figure Legend Snippet: METTL3 regulates JAK2 and SOCS3 expression via m 6 A methylation. a m 6 A dot blot analysis in piPSCs transfected with control, wild-type (WT), and mutant (MUT) METTL3 plasmid. Equal loading of mRNA was verified by methylene blue staining (lower panel). b Western blot analysis of METTL3, JAK2, and SOCS3 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. β-Actin was used as loading control. c AP staining of piPSCs transfected with control, WT, and MUT METTL3 plasmid. d Quantification of AP-positive colonies of piPSCs transfected with control, WT, and MUT METTL3 plasmid. e qPCR analysis of piPSCs transfected with control, WT, and MUT METTL3 plasmid. GAPDH was used as an internal control. f Western blot analysis of KLF4 and SOX2 in piPSCs transfected with control, WT, and MUT METTL3 plasmid. g Top consensus motif identified by HOMER with m 6 A-seq peaks in piPSCs. h Distribution of m 6 A peaks across the length of mRNA transcripts. Each region of 5′UTRs, CDSs, and 3′UTRs were binned into 100 segments, and the percentage of m 6 A peaks that fall within each bin was determined. i The m 6 A abundances in JAK2 and SOCS3 mRNA transcripts in piPSCs as detected by m 6 A-seq. The m 6 A peaks were shown in the black rectangles. j Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs with or without METTL3 knockdown. k MeRIP-qPCR analysis of m 6 A levels of JAK2 and SOCS3 in piPSCs transfected with control or METTL3 plasmid. l Relative luciferase activity of WT or MUT (A-to-T mutation) SOCS3-3′UTR (or JAK2-3′UTR) luciferase reporter in piPSCs transfected with control, WT, or MUT METTL3 plasmid. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data were presented as mean ± SD of three independent experiments. ** P

    Techniques Used: Expressing, Methylation, Dot Blot, Transfection, Mutagenesis, Plasmid Preparation, Staining, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Luciferase, Activity Assay

    11) Product Images from "Developmentally Regulated Recruitment of Transcription Factors and Chromatin Modification Activities to Chicken Lysozyme cis-Regulatory Elements In Vivo"

    Article Title: Developmentally Regulated Recruitment of Transcription Factors and Chromatin Modification Activities to Chicken Lysozyme cis-Regulatory Elements In Vivo

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.12.4386-4400.2003

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against CBP in HD50 MEP, HD11 and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07 kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against CBP in HD50 MEP, HD11 and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07 kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against C/EBPβ (NF-M) in HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against C/EBPβ (NF-M) in HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against Fli-1 (A) or NF1 (B) in HD50 MEP, HD37, HD11, and LPS-treated HD11 cells. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The hatched bars in the lower panel refer to a control experiment in HD50 MEP cells in which chromatin was not cross-linked. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining transcription factor binding to the lysozyme 5′ regulatory region with antibodies against Fli-1 (A) or NF1 (B) in HD50 MEP, HD37, HD11, and LPS-treated HD11 cells. The relative enrichment was calculated by normalizing the PCR signals to that of the −10.07-kb primer and the β-actin control primer as described in the text. The hatched bars in the lower panel refer to a control experiment in HD50 MEP cells in which chromatin was not cross-linked. The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    Chromatin immunoprecipitation assay examining the histone H3 modification status in HD37, HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. (A) Chromatin immunoprecipitation assay with antibodies recognizing histone H3 lysine 9 acetylation. The relative enrichment was calculated by normalizing the PCR signals to that of a β-actin control primer as described in the text. Note therefore that the ratio of β-actin to −10.07-kb signal intensity is constant. (B) Chromatin immunoprecipitation assay with antibodies recognizing methylated histone H3 lysine 9. In this case the relative enrichment or depletion was calculated by normalizing the PCR signals to that of the −10.07-kb primer as described in the text. The levels of histone H3 methylation at the boundaries of the DNase I-sensitive domain at −10.07 kb in the different cell lines are the same, as determined by normalization to signals obtained with the β-actin primer. The β-actin primer yielded signals that were two- to threefold lower that those obtained with the −10.07-kb primer (data not shown). The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.
    Figure Legend Snippet: Chromatin immunoprecipitation assay examining the histone H3 modification status in HD37, HD50 MEP, HD11, and LPS-treated HD11 cells as indicated. (A) Chromatin immunoprecipitation assay with antibodies recognizing histone H3 lysine 9 acetylation. The relative enrichment was calculated by normalizing the PCR signals to that of a β-actin control primer as described in the text. Note therefore that the ratio of β-actin to −10.07-kb signal intensity is constant. (B) Chromatin immunoprecipitation assay with antibodies recognizing methylated histone H3 lysine 9. In this case the relative enrichment or depletion was calculated by normalizing the PCR signals to that of the −10.07-kb primer as described in the text. The levels of histone H3 methylation at the boundaries of the DNase I-sensitive domain at −10.07 kb in the different cell lines are the same, as determined by normalization to signals obtained with the β-actin primer. The β-actin primer yielded signals that were two- to threefold lower that those obtained with the −10.07-kb primer (data not shown). The data are plotted as mean value of at least two independent chromatin immunoprecipitation assays and three independent amplifications.

    Techniques Used: Chromatin Immunoprecipitation, Modification, Polymerase Chain Reaction, Methylation

    12) Product Images from "Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies"

    Article Title: Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.15.10023-10031.2005

    Immunoprecipitation of NS2 complexes. (A) 293T cells were transfected with plasmids encoding NS2 and NS2f and labeled with [ 35 S]methionine. Proteins were immunoprecipitated with an anti-NS2 serum (lanes 1 through 4) or an anti-FLAG antibody (lanes 5 through
    Figure Legend Snippet: Immunoprecipitation of NS2 complexes. (A) 293T cells were transfected with plasmids encoding NS2 and NS2f and labeled with [ 35 S]methionine. Proteins were immunoprecipitated with an anti-NS2 serum (lanes 1 through 4) or an anti-FLAG antibody (lanes 5 through

    Techniques Used: Immunoprecipitation, Transfection, Labeling

    13) Product Images from "VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * S⃞"

    Article Title: VCP Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * Mutations Causing Frontotemporal Lobar Degeneration Disrupt Localization of TDP-43 and Induce Cell Death * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M900992200

    TDP-43 complexes with VCP in co-immunoprecipitation assays in both SHSY-5Y cells and human brain. A , TDP-43 immunoprecipitation ( IP ) in VCP-transfected SHSY-5Y cells probed with anti-VCP ( top ) and 5% loading control ( bottom ). B , VCP immunoprecipitation in high salt ( HS ) fraction of human brain probed with anti-TDP-43. Samples were immunoprecipitated with VCP: normal adult control cases ( NL-1 and NL-2 ); Alzheimer disease ( AD ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with the VCP R155H mutation ( VCP ) ( top ) and 2% loading control ( bottom ). C , TDP-43 immunoprecipitation in HS of human brain probed with anti-VCP: age-matched control ( NL-1 ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with VCP R155H mutation ( VCP ).
    Figure Legend Snippet: TDP-43 complexes with VCP in co-immunoprecipitation assays in both SHSY-5Y cells and human brain. A , TDP-43 immunoprecipitation ( IP ) in VCP-transfected SHSY-5Y cells probed with anti-VCP ( top ) and 5% loading control ( bottom ). B , VCP immunoprecipitation in high salt ( HS ) fraction of human brain probed with anti-TDP-43. Samples were immunoprecipitated with VCP: normal adult control cases ( NL-1 and NL-2 ); Alzheimer disease ( AD ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with the VCP R155H mutation ( VCP ) ( top ) and 2% loading control ( bottom ). C , TDP-43 immunoprecipitation in HS of human brain probed with anti-VCP: age-matched control ( NL-1 ); familial FTLD-U with GRN A9D mutation ( GRN ); sporadic ( FTLD-U ); and FTLD-U with VCP R155H mutation ( VCP ).

    Techniques Used: Immunoprecipitation, Transfection, Mutagenesis

    14) Product Images from "Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers"

    Article Title: Amplification of CRKL induces transformation and EGFR inhibitor resistance in human non small cell lung cancers

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-11-0046

    CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p
    Figure Legend Snippet: CRKL-induced cell transformation requires SOS1-RAS-RAF (A) Overexpression of CRKL increased RAS activity. The levels of GTP-bound RAS in AALE cells overexpressing a control vector or CRKL were measured by a pull-down assay followed by immunoblotting for RAS. Total RAS levels in total lysates were used as loading control. Positive and negative technical controls were obtained by incubating the total lysates with non-hydrolyzable analog of GTP (GTPγS) or GDP, respectively, before pull-down assays. (B) Overexpression of CRKL increased in vitro BRAF kinase activity. The BRAF proteins in AALE cells expressing indicated constructs were isolated by immunoprecipitation. The kinase activity was assessed by incubating with substrate proteins (MEK1). Immunoblots of phospho-MEK1 and BRAF proteins in the isolated BRAF immune complexes after kinase activity assay are shown. (C) Immunoblot of phospho-S338-RAF1 in AALE cell lines overexpressing wildtype or mutant CRKL. (D) Interaction between CRKL and SOS1 in AALE cells overexpressing CRKL. CRKL immune complexes were isolated followed by immunoblotting for SOS1 or CRKL proteins in AALE cells expressing indicated constructs. (E) CRKL-induced anchorage independent growth required SOS1-RAS-BRAF/RAF1 signaling. Left, Immunoblots of SOS1, KRAS, BRAF, RAF1 or ARAF proteins in CRKL-overexpressing AALE cell lines expressing a control shRNA targeting GFP or each gene-specific shRNA. ShRNAs that suppressed more than 50% of target protein levels were marked in red color. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p

    Techniques Used: Transformation Assay, Over Expression, Activity Assay, Plasmid Preparation, Pull Down Assay, In Vitro, Expressing, Construct, Isolation, Immunoprecipitation, Western Blot, Kinase Assay, Mutagenesis, shRNA

    15) Product Images from "Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families"

    Article Title: Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2018.05.003

    LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.
    Figure Legend Snippet: LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing, Negative Control, Incubation, Ligation, Amplification

    ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.
    Figure Legend Snippet: ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing

    LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.
    Figure Legend Snippet: LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.

    Techniques Used: Clone Assay, Plasmid Preparation, Expressing, Autoradiography, Labeling, Immunoprecipitation

    16) Product Images from "Rac1 Protein Rescues Neurite Retraction Caused by G2019S Leucine-rich Repeat Kinase 2 (LRRK2)"

    Article Title: Rac1 Protein Rescues Neurite Retraction Caused by G2019S Leucine-rich Repeat Kinase 2 (LRRK2)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.234005

    Co-immunoprecipitation of LRRK2 and Rac1. A , LRRK2 (V5, WT) was co-expressed with Rac1 (Myc, WT, CA, DN) in HEK293 FT cells, and LRRK2 was immunoprecipitated. Rac1 was readily detectable in complex. B , Co-association of LRRK2 and Rac1 was also readily apparent following overexpression of LRRK2 (WT) and Rac1 (CA) followed by immunoprecipitation ( IP ) of Rac1 and immunoblotting ( IB ) for LRRK2. C , association of LRRK2 with Rho GTPases appears to be strongest for Rac1. LRRK2 (V5, WT) was co-expressed with Rac1, RhoA, or Cdc42 (Myc, WT for each) in HEK293 cells, and the LRRK2 was immunoprecipitated. Rac1 and Cdc42 were detectable in the complexes following a rank order for detection of Rac1 > Cdc42 ≫ RhoA. D , LRRK2 was immunoprecipitated from human brain striatal lysate and then probed with anti-Rac1 antibody. A robust Rac1 signal was evident in the immunoprecipitate but absent when nonspecific IgG was substituted for the LRRK2 antibody. E , LRRK2 (GFP, WT) deletion constructs were expressed with Rac1 (Myc, CA) to identify which domains are most important for binding. The LRRK2 was immunoprecipitated and immunoblotted for Rac1. Rac1 binding was apparent with the ROC-COR kinase constructs, as well as with constructs carrying the subdomains of COR or kinase. KIN , kinase; RCK , ROC-COR-kinase domains; CoIP , co-immunoprecipitation.
    Figure Legend Snippet: Co-immunoprecipitation of LRRK2 and Rac1. A , LRRK2 (V5, WT) was co-expressed with Rac1 (Myc, WT, CA, DN) in HEK293 FT cells, and LRRK2 was immunoprecipitated. Rac1 was readily detectable in complex. B , Co-association of LRRK2 and Rac1 was also readily apparent following overexpression of LRRK2 (WT) and Rac1 (CA) followed by immunoprecipitation ( IP ) of Rac1 and immunoblotting ( IB ) for LRRK2. C , association of LRRK2 with Rho GTPases appears to be strongest for Rac1. LRRK2 (V5, WT) was co-expressed with Rac1, RhoA, or Cdc42 (Myc, WT for each) in HEK293 cells, and the LRRK2 was immunoprecipitated. Rac1 and Cdc42 were detectable in the complexes following a rank order for detection of Rac1 > Cdc42 ≫ RhoA. D , LRRK2 was immunoprecipitated from human brain striatal lysate and then probed with anti-Rac1 antibody. A robust Rac1 signal was evident in the immunoprecipitate but absent when nonspecific IgG was substituted for the LRRK2 antibody. E , LRRK2 (GFP, WT) deletion constructs were expressed with Rac1 (Myc, CA) to identify which domains are most important for binding. The LRRK2 was immunoprecipitated and immunoblotted for Rac1. Rac1 binding was apparent with the ROC-COR kinase constructs, as well as with constructs carrying the subdomains of COR or kinase. KIN , kinase; RCK , ROC-COR-kinase domains; CoIP , co-immunoprecipitation.

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

    Immunoprecipitation of LRRK2 constructs carrying disease-linked mutations. G2019S and R1441C exhibit weaker binding to CARac1, whereas Y1699C and I2020T exhibit increased binding. Shown is a representative immunoblot ( IB ). Shown is a quantification of three independent experiments ( n = 3). *, p
    Figure Legend Snippet: Immunoprecipitation of LRRK2 constructs carrying disease-linked mutations. G2019S and R1441C exhibit weaker binding to CARac1, whereas Y1699C and I2020T exhibit increased binding. Shown is a representative immunoblot ( IB ). Shown is a quantification of three independent experiments ( n = 3). *, p

    Techniques Used: Immunoprecipitation, Construct, Binding Assay

    Knockdown of LRRK2 by siRNA reduces neurite process length. A and B , siRNA-mediated knockdown of LRRK2 reduced LRRK2 protein levels in HEK293 FT cells. C and D , fluorescent tagged siRNA was used to knockdown LRRK2 in differentiated SH-SY5Y cells. IP , immunoprecipitation; IB , immunoblot. Neg Ctrl , negative control.
    Figure Legend Snippet: Knockdown of LRRK2 by siRNA reduces neurite process length. A and B , siRNA-mediated knockdown of LRRK2 reduced LRRK2 protein levels in HEK293 FT cells. C and D , fluorescent tagged siRNA was used to knockdown LRRK2 in differentiated SH-SY5Y cells. IP , immunoprecipitation; IB , immunoblot. Neg Ctrl , negative control.

    Techniques Used: Immunoprecipitation, Negative Control

    17) Product Images from "Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway"

    Article Title: Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007882

    IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).
    Figure Legend Snippet: IE4 interacts with ASF/SF2, 9G8 and SRp20 through its arginine-rich Ra, Rb and Rc domains. (A) Nuclear extracts from co-transfected HeLa cells were immunoprecipitated with anti-GFP, anti-V5 or anti-HA antibodies as indicated. In Input (lanes 1 and 7), nuclear extracts were fractionated without immunoprecipitation. Nuclear extracts were treated (lane 6) or not (lane 5) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. In lanes 3 and 9, immunoprecipitation was performed with beads without antibody as a negative control (−). The blots were probed with anti-HA (left panels) or anti-V5 (right panels) antibodies. (B) Total extracts from transfected HeLa cells were immunoprecipitated with anti-Flag or anti-V5 antibodies as indicated. In Input (lanes 1, 2, 5 and 6), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-ASF/SF2 or anti-SRp20 (upper panels) and anti-V5 (lower panels) antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (C) In vitro binding assay was performed by incubating GST-ASF/SF2 with in vitro -translated IE4-V5 or derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (−) (lane 1). In Input (lanes 8 to 14), [ 35 S]-methionine-labelled proteins were fractionated without binding assay. (D) In vitro -translated ASF/SF2-V5 was incubated with GST-IE4 derivatives as indicated (lanes 2 to 7). GST alone constituted the negative control (lane 1) and, in Input, ASF/SF2-V5 was loaded without binding assay (lane 8).

    Techniques Used: Transfection, Immunoprecipitation, Negative Control, In Vitro, Binding Assay, Incubation

    IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.
    Figure Legend Snippet: IE4 interacts with SRPK1 through its arginine-rich Ra and Rb domains. (A) Total extracts of transfected and mock- or VZV-infected MeWo cells were immunoprecipitated with anti-GFP, anti-IE4, anti-V5 or anti-Flag antibodies as indicated (lanes 3 to 6). In Input (lanes 1 and 2), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Flag or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (B) Binding assay was performed by incubating HEK293 cells total extracts with GST-IE4 derivatives as indicated (lanes 2 to 7). Assay performed with GST alone constituted the negative control (lane 1). The blot was probed with anti-SRPK1 antibody. Coomassie Blue-stained gel is shown below.

    Techniques Used: Transfection, Infection, Immunoprecipitation, Binding Assay, Negative Control, Staining

    IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.
    Figure Legend Snippet: IE4 interacts with TAP/NXF1 and Aly/REF. (A) In vitro binding assays were performed by incubating GST-TAP (lanes 2 to 7) or GST-REF (lanes 9 to 14) with in vitro -translated IE4-V5 or derivatives as indicated. Assays performed with GST alone constituted the negative control (−) (lanes 1 and 8). (B) In vitro -translated IE4 Nter-V5 was incubated with GST-TAP (lanes 4 and 5) or GST-REF (lanes 6 and 7). GST alone constituted the negative control (lanes 2 and 3). Complexes were treated (lanes 3, 5 and 7) or not (lanes 2, 4 and 6) with RNases A/T1 mix for 30 min at 37°C before SDS-PAGE. In Input (lane 1), IE4 Nter-V5 was loaded without binding assay. (C) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lane 2), anti-IE4 (lane 3), anti-Flag (lane 5), anti-Aly/REF or anti-TAP/NXF1 (lane 6) antibodies as indicated. In Input (lanes 1 and 4), total extracts were fractionated without immunoprecipitation. The blots were probed with anti-Aly/REF, anti-TAP/NXF1 or anti-IE4 antibodies. The asterisk marks heavy chain IgG from the immunoprecipitation. (D) Total extracts of VZV-infected MeWo cells were immunoprecipitated with anti-GFP (lanes 3 and 8), anti-Aly/REF (lanes 4 and 5) or anti-TAP/NXF1 (lanes 9 and 10) antibodies as indicated. In Input (lanes 1, 2, 6 and 7), total extracts were fractionated without immunoprecipitation. Total extracts were treated (lanes 5 and 10) or not (lanes 3, 4, 8 and 9) with RNases A/T1 mix for 30 min at 37°C before immunoprecipitation. The blots were probed with anti-IE4, anti-Aly/REF or anti-TAP/NXF1 antibodies.

    Techniques Used: In Vitro, Binding Assay, Negative Control, Incubation, SDS Page, Infection, Immunoprecipitation

    18) Product Images from "Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells"

    Article Title: Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2015.10.012

    Co-immunoprecipitation (co-IP) experiments in HeLa cells. Ubiquitinated SMN protein was determined by co-IP using anti-SMN antibody. (A) Ubiquitinated SMN extracted by co-IP. HeLa cells were incubated with salbutamol sulfate (20 µM) for 24 h in the presence or absence of the proteasome inhibitor, MG132 (5 µM). SMN ubiquitination was reduced in HeLa cells treated with salbutamol, in the presence or absence of MG132. (B) Quantification of ubiquitinated SMN levels. The amount of ubiquitinated SMN at the mock status (not treated with salbutamol) was normalized to 1.0 in each MG132 group (in the presence or absence of MG132). The mean ubiquitinated SMN amount decreased by 20–30 percent after salbutamol treatment.
    Figure Legend Snippet: Co-immunoprecipitation (co-IP) experiments in HeLa cells. Ubiquitinated SMN protein was determined by co-IP using anti-SMN antibody. (A) Ubiquitinated SMN extracted by co-IP. HeLa cells were incubated with salbutamol sulfate (20 µM) for 24 h in the presence or absence of the proteasome inhibitor, MG132 (5 µM). SMN ubiquitination was reduced in HeLa cells treated with salbutamol, in the presence or absence of MG132. (B) Quantification of ubiquitinated SMN levels. The amount of ubiquitinated SMN at the mock status (not treated with salbutamol) was normalized to 1.0 in each MG132 group (in the presence or absence of MG132). The mean ubiquitinated SMN amount decreased by 20–30 percent after salbutamol treatment.

    Techniques Used: Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation

    19) Product Images from "Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab"

    Article Title: Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab

    Journal: Journal of Virology

    doi:

    Detection of ORF 1a-encoded 3CL pro cleavage products in HCV 229E-infected cells. (A) Metabolically labeled lysates from mock-infected (M) (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or HCV 229E-infected (I) (lanes 2, 4, 6, 8, 10, 12, 14, and 16) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with the pp1a-specific rabbit antisera α-p5, α-p23, α-p12, and α-p16 or the corresponding preimmune sera. The cells were labeled from 5 to 12 h p.i. Either 180 μl (lanes 1 to 4) or 70 μl (lanes 5 to 16) was analyzed after immunoprecipitation with preimmune serum (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or with the appropriate antiserum as indicated (lanes 3, 4, 7, 8, 11, 12, 15, and 16). (B) Metabolically labeled lysates (100 μl) from mock-infected (lanes 1 and 2) or HCV 229E-infected (lanes 3 and 4) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with antiserum α-p16 (lanes 2 and 4) or the corresponding preimmune serum (lanes 1 and 3). The cells were labeled from 5 to 12 h p.i. Sizes of protein molecular mass markers (lanes PM) (CFA626 and CFA645; Amersham Pharmacia Biotech) and the processing products p5, p12, p23, and p16 are indicated.
    Figure Legend Snippet: Detection of ORF 1a-encoded 3CL pro cleavage products in HCV 229E-infected cells. (A) Metabolically labeled lysates from mock-infected (M) (lanes 1, 3, 5, 7, 9, 11, 13, and 15) or HCV 229E-infected (I) (lanes 2, 4, 6, 8, 10, 12, 14, and 16) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with the pp1a-specific rabbit antisera α-p5, α-p23, α-p12, and α-p16 or the corresponding preimmune sera. The cells were labeled from 5 to 12 h p.i. Either 180 μl (lanes 1 to 4) or 70 μl (lanes 5 to 16) was analyzed after immunoprecipitation with preimmune serum (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or with the appropriate antiserum as indicated (lanes 3, 4, 7, 8, 11, 12, 15, and 16). (B) Metabolically labeled lysates (100 μl) from mock-infected (lanes 1 and 2) or HCV 229E-infected (lanes 3 and 4) MRC-5 cells were analyzed by SDS–17.4% polyacrylamide gel electrophoresis after immunoprecipitation with antiserum α-p16 (lanes 2 and 4) or the corresponding preimmune serum (lanes 1 and 3). The cells were labeled from 5 to 12 h p.i. Sizes of protein molecular mass markers (lanes PM) (CFA626 and CFA645; Amersham Pharmacia Biotech) and the processing products p5, p12, p23, and p16 are indicated.

    Techniques Used: Infection, Metabolic Labelling, Labeling, Polyacrylamide Gel Electrophoresis, Immunoprecipitation

    20) Product Images from "Human Cytomegalovirus IE2 86 and IE2 40 Proteins Differentially Regulate UL84 Protein Expression Posttranscriptionally in the Absence of Other Viral Gene Products ▿"

    Article Title: Human Cytomegalovirus IE2 86 and IE2 40 Proteins Differentially Regulate UL84 Protein Expression Posttranscriptionally in the Absence of Other Viral Gene Products ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00090-10

    The first 105 aa of UL84 are important for interaction with IE2 86 and IE2 40. wt UL84 and each of the N-terminal mutants, Δ68 (68) and Δ105 (105), were assayed for their interaction with both IE2 86 and IE2 40 using immunoprecipitation
    Figure Legend Snippet: The first 105 aa of UL84 are important for interaction with IE2 86 and IE2 40. wt UL84 and each of the N-terminal mutants, Δ68 (68) and Δ105 (105), were assayed for their interaction with both IE2 86 and IE2 40 using immunoprecipitation

    Techniques Used: Immunoprecipitation

    21) Product Images from "SPBP Is a Phosphoserine-Specific Repressor of Estrogen Receptor ?"

    Article Title: SPBP Is a Phosphoserine-Specific Repressor of Estrogen Receptor ?

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.9.3421-3430.2005

    Interaction of endogenous SPBP and ERα. (A) Coimmunoprecipitation of endogenous ERα and SPBP. Extracts were prepared from confluent cultures of MCF7-SH cells, stimulated where indicated with E2 for 3 h. Immunoprecipitation (IP) was performed
    Figure Legend Snippet: Interaction of endogenous SPBP and ERα. (A) Coimmunoprecipitation of endogenous ERα and SPBP. Extracts were prepared from confluent cultures of MCF7-SH cells, stimulated where indicated with E2 for 3 h. Immunoprecipitation (IP) was performed

    Techniques Used: Immunoprecipitation

    22) Product Images from "West Nile Virus NS1 Antagonizes Interferon Beta Production by Targeting RIG-I and MDA5"

    Article Title: West Nile Virus NS1 Antagonizes Interferon Beta Production by Targeting RIG-I and MDA5

    Journal: Journal of Virology

    doi: 10.1128/JVI.02396-16

    Analysis of the interaction between NS1 and RIG-I/MDA5 by co-IP assay. HEK-293T cells were cotransfected with plasmid expressing HA-tagged NS1 and a FLAG-tagged RIG-I/MDA5 expression plasmid or an empty vector for 36 h. The cells were lysed and subjected to immunoprecipitation with rabbit anti-HA or mouse anti-FLAG Ab. Rabbit or mouse normal IgG was used as the negative control. The IP products and input samples were analyzed by immunoblotting using mouse anti-HA and mouse anti-FLAG Abs. One representative experiment out of three is shown. WB, Western blot.
    Figure Legend Snippet: Analysis of the interaction between NS1 and RIG-I/MDA5 by co-IP assay. HEK-293T cells were cotransfected with plasmid expressing HA-tagged NS1 and a FLAG-tagged RIG-I/MDA5 expression plasmid or an empty vector for 36 h. The cells were lysed and subjected to immunoprecipitation with rabbit anti-HA or mouse anti-FLAG Ab. Rabbit or mouse normal IgG was used as the negative control. The IP products and input samples were analyzed by immunoblotting using mouse anti-HA and mouse anti-FLAG Abs. One representative experiment out of three is shown. WB, Western blot.

    Techniques Used: Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, Immunoprecipitation, Negative Control, Western Blot

    23) Product Images from "Role of NH2- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication"

    Article Title: Role of NH2- and COOH-Terminal Domains of the P Protein of Human Parainfluenza Virus Type 3 in Transcription and Replication

    Journal: Journal of Virology

    doi:

    Analysis of P-P and L-P complexes in a flag-tagged immunoprecipitation system. The proteins were coexpressed and metabolically labeled with [ 35 S]methionine. The labeled proteins present in the cytosolic fraction were immunoprecipitated with anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS–10% polyacrylamide gel followed by fluorography. (A) Analysis of P-P complex; 200 ng of flag-tagged P (P f ) plasmid DNA was used in the transfection. Numbers above the lanes indicate the amount of untagged mutant (P mut ) and wt P protein-expressing plasmid DNA (in micrograms) used for transfection. The ratios of untagged to flag-tagged P proteins are shown below. (B) Analysis of L-P complex; 200 ng of flag-tagged L-expressing plasmid DNA (L f ) was used in all transfections. The untagged wt and mutant P protein-expressing plasmid DNAs, where indicated, were used at a concentration of 1 μg each. The ratios of untagged P protein to flag-tagged L in each case are shown below. Radiolabeled bands were visualized by fluorography and quantitated by phosphorimager.
    Figure Legend Snippet: Analysis of P-P and L-P complexes in a flag-tagged immunoprecipitation system. The proteins were coexpressed and metabolically labeled with [ 35 S]methionine. The labeled proteins present in the cytosolic fraction were immunoprecipitated with anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS–10% polyacrylamide gel followed by fluorography. (A) Analysis of P-P complex; 200 ng of flag-tagged P (P f ) plasmid DNA was used in the transfection. Numbers above the lanes indicate the amount of untagged mutant (P mut ) and wt P protein-expressing plasmid DNA (in micrograms) used for transfection. The ratios of untagged to flag-tagged P proteins are shown below. (B) Analysis of L-P complex; 200 ng of flag-tagged L-expressing plasmid DNA (L f ) was used in all transfections. The untagged wt and mutant P protein-expressing plasmid DNAs, where indicated, were used at a concentration of 1 μg each. The ratios of untagged P protein to flag-tagged L in each case are shown below. Radiolabeled bands were visualized by fluorography and quantitated by phosphorimager.

    Techniques Used: Immunoprecipitation, Metabolic Labelling, Labeling, Plasmid Preparation, Transfection, Mutagenesis, Expressing, Concentration Assay

    Stoichiometric requirement for P for efficient minigenome replication, and role of terminal domains in this process. (A) The complete minigenome replication system contained pHPIV3MG(−) (200 ng), pN (640 ng), pLf (200 ng), and pP at a suboptimal concentration (300 ng). pP, pPΔ40N, and pPΔ20C (patterned bars from left to right) were expressed, in addition to the components present in the complete system (solid bar), as indicated above, in increasing concentrations (300, 600, and 900 ng), and luciferase activity in the cell extract was determined. (B) Western blot analysis of N and P proteins using anti-RNP antibody that recognized the N and P proteins. The migration positions of N and P are shown. A protein band migrating faster than N may be a degradation product of PΔ40N. The amounts of plasmid DNAs used are equivalent to the amounts indicated for the complete system. In addition, lanes P wt , P Δ40N , and P Δ20C contained 900 ng of the respective plasmid DNAs for transfection. (C) Immunoprecipitation of radiolabeled proteins from cells that were transfected with plasmid DNAs as described for panel B, except the first lane contained pLf alone. Cells were metabolically labeled with [ 35 S]methionine and immunoprecipitated by using anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS–10% polyacrylamide gel and subjected to fluorography. The data are representative of three separate experiments with an experimental variability of
    Figure Legend Snippet: Stoichiometric requirement for P for efficient minigenome replication, and role of terminal domains in this process. (A) The complete minigenome replication system contained pHPIV3MG(−) (200 ng), pN (640 ng), pLf (200 ng), and pP at a suboptimal concentration (300 ng). pP, pPΔ40N, and pPΔ20C (patterned bars from left to right) were expressed, in addition to the components present in the complete system (solid bar), as indicated above, in increasing concentrations (300, 600, and 900 ng), and luciferase activity in the cell extract was determined. (B) Western blot analysis of N and P proteins using anti-RNP antibody that recognized the N and P proteins. The migration positions of N and P are shown. A protein band migrating faster than N may be a degradation product of PΔ40N. The amounts of plasmid DNAs used are equivalent to the amounts indicated for the complete system. In addition, lanes P wt , P Δ40N , and P Δ20C contained 900 ng of the respective plasmid DNAs for transfection. (C) Immunoprecipitation of radiolabeled proteins from cells that were transfected with plasmid DNAs as described for panel B, except the first lane contained pLf alone. Cells were metabolically labeled with [ 35 S]methionine and immunoprecipitated by using anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS–10% polyacrylamide gel and subjected to fluorography. The data are representative of three separate experiments with an experimental variability of

    Techniques Used: Concentration Assay, Luciferase, Activity Assay, Western Blot, Migration, Plasmid Preparation, Transfection, Immunoprecipitation, Metabolic Labelling, Labeling

    24) Product Images from "Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction"

    Article Title: Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.06.022

    Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).
    Figure Legend Snippet: Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).

    Techniques Used: Western Blot, Quantitation Assay, Expressing, Immunofluorescence, Cell Culture, Immunoprecipitation, Transfection, Binding Assay, Purification, Overlay Assay

    25) Product Images from "The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex"

    Article Title: The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex

    Journal: Cancers

    doi: 10.3390/cancers11121974

    Exogenous Syk expression increases E-cadherin and catenin phosphorylation and interaction. ( a ) Representative image of MCF7 cells that stably express GFP-Syk. Scale bar: 200 μm. Whole cell lysates (WCL) of parental and GFP-Syk MCF7 cells were analyzed by Western blotting (WB) with anti-Syk, -E-Cdh, -α-, -β-, and -p120-Ctn antibodies. β-tubulin was used as loading control. ( b ) Protein lysates of nontransfected and GFP-Syk-expressing MCF7 cells were immunoprecipitated (IP) with anti-E-Cdh, -α- and -β-Ctn antibodies and analyzed by Western blotting to detect E-Cdh/Ctn phosphorylation at the indicated tyrosine residues. ( c ) Protein lysates of HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were immunoprecipitated (IP) using anti-E-Cdh, -α- and -β-Ctn or -Syk antibodies and analyzed by Western blotting (WB). ( d – f ) E-cadherin and catenin interaction was evaluated by co-immunoprecipitation (IP) followed by Western blotting (WB) using the indicated antibodies against E-Cdh, α-, and β-Ctn and the different phosphorylated forms and protein lysates from parental MCF7 cells (d), nontransfected and GFP-Syk-expressing MCF7 cells (e), and HEK293T cells transiently transfected or not with a FLAG-Syk plasmid (f).
    Figure Legend Snippet: Exogenous Syk expression increases E-cadherin and catenin phosphorylation and interaction. ( a ) Representative image of MCF7 cells that stably express GFP-Syk. Scale bar: 200 μm. Whole cell lysates (WCL) of parental and GFP-Syk MCF7 cells were analyzed by Western blotting (WB) with anti-Syk, -E-Cdh, -α-, -β-, and -p120-Ctn antibodies. β-tubulin was used as loading control. ( b ) Protein lysates of nontransfected and GFP-Syk-expressing MCF7 cells were immunoprecipitated (IP) with anti-E-Cdh, -α- and -β-Ctn antibodies and analyzed by Western blotting to detect E-Cdh/Ctn phosphorylation at the indicated tyrosine residues. ( c ) Protein lysates of HEK293T cells transiently transfected or not with a FLAG-Syk plasmid were immunoprecipitated (IP) using anti-E-Cdh, -α- and -β-Ctn or -Syk antibodies and analyzed by Western blotting (WB). ( d – f ) E-cadherin and catenin interaction was evaluated by co-immunoprecipitation (IP) followed by Western blotting (WB) using the indicated antibodies against E-Cdh, α-, and β-Ctn and the different phosphorylated forms and protein lysates from parental MCF7 cells (d), nontransfected and GFP-Syk-expressing MCF7 cells (e), and HEK293T cells transiently transfected or not with a FLAG-Syk plasmid (f).

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

    26) Product Images from "WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W] [OA]"

    Article Title: WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.109.143404

    WPP domain proteins WPP1 and WPP2 interact with both WIT1 and HSC70 in vivo. A, HA-HSC70-1 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (left); HA-HSC70-3 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (right) in N. benthamiana . Immunoprecipitation
    Figure Legend Snippet: WPP domain proteins WPP1 and WPP2 interact with both WIT1 and HSC70 in vivo. A, HA-HSC70-1 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (left); HA-HSC70-3 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (right) in N. benthamiana . Immunoprecipitation

    Techniques Used: In Vivo, Immunoprecipitation

    27) Product Images from "WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W] [OA]"

    Article Title: WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W]WPP-Domain Proteins Mimic the Activity of the HSC70-1 Chaperone in Preventing Mistargeting of RanGAP1-Anchoring Protein WIT1 1 [C] [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.109.143404

    WPP domain proteins WPP1 and WPP2 interact with both WIT1 and HSC70 in vivo. A, HA-HSC70-1 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (left); HA-HSC70-3 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (right) in N. benthamiana . Immunoprecipitation
    Figure Legend Snippet: WPP domain proteins WPP1 and WPP2 interact with both WIT1 and HSC70 in vivo. A, HA-HSC70-1 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (left); HA-HSC70-3 was coexpressed with either GFP, WPP1-GFP, or WPP2-GFP (right) in N. benthamiana . Immunoprecipitation

    Techniques Used: In Vivo, Immunoprecipitation

    28) Product Images from "Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC"

    Article Title: Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00145.2018

    Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A : representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) immunoprecipitation (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left : blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1 : H 2 0-injected oocytes; lane 2 : WNK4-injected oocytes; lane 3 : WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4 : WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5 : WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right : blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1 : IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2 : flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3 : IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4 : flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left : blot from total lysate shows total WNK4 and pWNK4, as stated. Right : blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1 , 3 , and 5 were loaded with proteins from the flowthrough, whereas lanes 2 , 4 , and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.
    Figure Legend Snippet: Kidney-specific with no lysine kinase 1 (KS-WNK1) promotes WNK4 S335 phosphorylation through heterodimer formation. A : representative Western blot of the total lysate and KS-WNK1 lacking exon 11 (KS-WNK1-Δ11) immunoprecipitation (IP) from oocytes injected with WNK4 cRNA alone or together with KS-WNK1-Δ11 cRNA or KS-WNK1-Δ11 HQ/AA, as stated. IP was performed using the c-Myc tag antibody, which exclusively recognizes KS-WNK1-Δ11. Left : blot from total lysate shows total WNK4 and pWNK4 expression, as stated. Lanes were loaded as follows: lane 1 : H 2 0-injected oocytes; lane 2 : WNK4-injected oocytes; lane 3 : WNK4-injected oocytes exposed to low-chloride hypotonic stress (LCHS) conditions; lane 4 : WNK4 + KS-WNK1-Δ11-injected oocytes; lane 5 : WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Right : blot from the IP assay. IP was performed against the c-Myc tag, which recognizes KS-WNK1-Δ11 and revealed against WNK4 or pWNK4, as stated. Lanes were loaded as follows: lane 1 : IP from WNK4 + KS-WNK1-Δ11-injected oocytes; lane 2 : flowthrough of WNK4 + KS-WNK1-Δ11-injected oocytes; lane 3 : IP from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes (the absence of WNK4 in the IP confirms the specificity of the c-Myc IP); lane 4 : flowthrough from WNK4 + KS-WNK1-Δ11 HQ/AA-injected oocytes. Identical results were observed in 3 independent experiments. B : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 L322F cRNA or WNK4 S335A alone or together with KS-WNK1-Δ11 cRNA, as stated. Left : blot from total lysate shows total WNK4 and pWNK4, as stated. Right : blot from the IP assay. Lanes 1 and 3 were loaded with proteins from the IP fraction, whereas lanes 2 and 4 were loaded with proteins from the flowthrough, as stated. The blot shows pWNK4 and WNK4 expression, as stated. Identical results were observed in 3 different experiments. C : representative Western blot of the total lysate and KS-WNK1-Δ11 IP from oocytes injected with WNK4 S335A cRNA together with KS-WNK1-Δ11 or KS-WNK1-Δ11-Δ4a cRNA, as stated. Blot from the IP assay is shown. Lanes 1 , 3 , and 5 were loaded with proteins from the flowthrough, whereas lanes 2 , 4 , and 6 were loaded with proteins from the IP fraction, as stated. The blot shows total WNK4 and pWNK4 expression, as stated. Identical results were obtained from 3 independent experiments.

    Techniques Used: Western Blot, Immunoprecipitation, Injection, Expressing

    29) Product Images from "The levels of H11/HspB8 DNA methylation in human melanoma tissues and xenografts are a critical molecular marker for 5'-Aza-2-deoxycytidine therapy"

    Article Title: The levels of H11/HspB8 DNA methylation in human melanoma tissues and xenografts are a critical molecular marker for 5'-Aza-2-deoxycytidine therapy

    Journal: Cancer investigation

    doi: 10.3109/07357907.2011.584588

    The H11/HspB8 mutant P173H does not bind TAK1 (A) Reciprocal pull-down assay of protein extracts from MeWo cells (express P173H) using antibodies to TAK1 (T), H11/HspB8 (H), or normal IgG (Ig) in immunoprecipitation (IP) and immunoblotting (IB). ( B ) Reciprocal pull-down assay of protein extracts from SKMEL-2 cells stably transfected with tet-regulated H11/HspB8 untreated or treated with Dox (5μg/ml; 3d) done as in (A) . Molecular weights are shown on the right. * indicates slower migrating phosphorylated TAK1 protein.
    Figure Legend Snippet: The H11/HspB8 mutant P173H does not bind TAK1 (A) Reciprocal pull-down assay of protein extracts from MeWo cells (express P173H) using antibodies to TAK1 (T), H11/HspB8 (H), or normal IgG (Ig) in immunoprecipitation (IP) and immunoblotting (IB). ( B ) Reciprocal pull-down assay of protein extracts from SKMEL-2 cells stably transfected with tet-regulated H11/HspB8 untreated or treated with Dox (5μg/ml; 3d) done as in (A) . Molecular weights are shown on the right. * indicates slower migrating phosphorylated TAK1 protein.

    Techniques Used: Mutagenesis, Pull Down Assay, Immunoprecipitation, Stable Transfection, Transfection

    H11/HspB8 methylation is inversely related to gene expression (A) Bisulfite treated DNA and mRNA isolated from cell suspensions of metastatic melanoma tissues, normal human melanocyte cultures (NHM) and melanoma cell lines SKMEL-2, MeWo and A2058 were assayed by MSP and QRT-PCR, respectively. MSP results are expressed as +/− based on the detection of methylated PCR products in 1.7% agarose gels. Data for QRT- PCR are expressed as ΔCt values calculated as described in Materials and Methods. Low ΔCt values indicate high expression. The ΔCt value for NHM (5.4) was used as cut-off point (dotted line) to assess the ΔCt values for the melanoma tissues and they are shown relative to the MSP results. ( B ) Bisulfite treated DNA from early passage melanoma cultures EK, NL, VO, RP, MS, LB, IH, and EI, established melanoma cell lines MeWo, A2058, A375, SKMEL2, LM and NHM was analyzed by MSQP and gene expression was determined by immunoprecipitation/immunoblotting with H11/ HspB8 antibody. Data are expressed as methylation and densitometric units, respectively. The inverse correlation between H11/HspB8 methylation and gene expression is statistically significant (p
    Figure Legend Snippet: H11/HspB8 methylation is inversely related to gene expression (A) Bisulfite treated DNA and mRNA isolated from cell suspensions of metastatic melanoma tissues, normal human melanocyte cultures (NHM) and melanoma cell lines SKMEL-2, MeWo and A2058 were assayed by MSP and QRT-PCR, respectively. MSP results are expressed as +/− based on the detection of methylated PCR products in 1.7% agarose gels. Data for QRT- PCR are expressed as ΔCt values calculated as described in Materials and Methods. Low ΔCt values indicate high expression. The ΔCt value for NHM (5.4) was used as cut-off point (dotted line) to assess the ΔCt values for the melanoma tissues and they are shown relative to the MSP results. ( B ) Bisulfite treated DNA from early passage melanoma cultures EK, NL, VO, RP, MS, LB, IH, and EI, established melanoma cell lines MeWo, A2058, A375, SKMEL2, LM and NHM was analyzed by MSQP and gene expression was determined by immunoprecipitation/immunoblotting with H11/ HspB8 antibody. Data are expressed as methylation and densitometric units, respectively. The inverse correlation between H11/HspB8 methylation and gene expression is statistically significant (p

    Techniques Used: Methylation, Expressing, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Mass Spectrometry, Immunoprecipitation

    30) Product Images from "Transforming Growth Factor β‐Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins"

    Article Title: Transforming Growth Factor β‐Activated Kinase 1 Regulates Mesenchymal Stem Cell Proliferation Through Stabilization of Yap1/Taz Proteins

    Journal: Stem Cells (Dayton, Ohio)

    doi: 10.1002/stem.3083

    Localization of TGFβ‐activated kinase 1 (Tak1) and interaction between Tak1‐Yap1/Taz. (A): Localization change of Tak1, Yap1, and Taz by 5zox treatment. Tbp is a TATA‐binding protein as a representative of nuclear localizing protein. αTubulin is a representative of cytoplasmic protein. (B): Immunoprecipitation (IP)‐Western blot (WB) showing interaction between Tak1 and Yap1/Taz. HA indicates IP fraction prepared with anti‐HA antibody. (C): IP‐WB showing affinity change between Tak1 and Yap1/Taz by 5zox treatment. IP fractions were prepared with anti‐HA antibody, which bound to HA‐Tak1. (D): Phosphorylation status of Yap1 and Tak1 under control and 5zox treatment condition. (E): IP‐based detection of ubiquitination status of Yap1 and Taz. Total Yap1 or Taz was collected from cell lysate of BMMSCs with anti‐HA antibody. Subsequently, the ubiquitinated Yap1 or Taz were detected with anti‐Ub antibody. Abbreviations: C, Cytoplasmic fraction; N, nuclear fraction of bone marrow‐derived mesenchymal stem cells (BMMSCs).
    Figure Legend Snippet: Localization of TGFβ‐activated kinase 1 (Tak1) and interaction between Tak1‐Yap1/Taz. (A): Localization change of Tak1, Yap1, and Taz by 5zox treatment. Tbp is a TATA‐binding protein as a representative of nuclear localizing protein. αTubulin is a representative of cytoplasmic protein. (B): Immunoprecipitation (IP)‐Western blot (WB) showing interaction between Tak1 and Yap1/Taz. HA indicates IP fraction prepared with anti‐HA antibody. (C): IP‐WB showing affinity change between Tak1 and Yap1/Taz by 5zox treatment. IP fractions were prepared with anti‐HA antibody, which bound to HA‐Tak1. (D): Phosphorylation status of Yap1 and Tak1 under control and 5zox treatment condition. (E): IP‐based detection of ubiquitination status of Yap1 and Taz. Total Yap1 or Taz was collected from cell lysate of BMMSCs with anti‐HA antibody. Subsequently, the ubiquitinated Yap1 or Taz were detected with anti‐Ub antibody. Abbreviations: C, Cytoplasmic fraction; N, nuclear fraction of bone marrow‐derived mesenchymal stem cells (BMMSCs).

    Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Derivative Assay

    31) Product Images from "Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae"

    Article Title: Phosphorylation by Cak1 Regulates the C-Terminal Domain Kinase Ctk1 in Saccharomyces cerevisiae

    Journal:

    doi: 10.1128/MCB.25.10.3906-3913.2005

    Thr-338 phosphorylation is required for Ctk1 activity. (A) Ctk1-HA was immunoprecipitated from yeast strains expressing vector (−HA), wild-type ( WT ) Ctk1, Ctk1 T338A , and Ctk1 D324A (lanes 1 to 4). The expression levels and efficiencies of immunoprecipitation
    Figure Legend Snippet: Thr-338 phosphorylation is required for Ctk1 activity. (A) Ctk1-HA was immunoprecipitated from yeast strains expressing vector (−HA), wild-type ( WT ) Ctk1, Ctk1 T338A , and Ctk1 D324A (lanes 1 to 4). The expression levels and efficiencies of immunoprecipitation

    Techniques Used: Activity Assay, Immunoprecipitation, Expressing, Plasmid Preparation

    32) Product Images from "Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families"

    Article Title: Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2018.05.003

    LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.
    Figure Legend Snippet: LOX forms a complex with ADAMTSL2 and ADAMTS10 A. Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing LOX plus ADAMTSL2, ADAMTSL2 alone (negative control), LOX plus ADAMTS10, or ADAMTS10 alone (negative control). B. Co-immunoprecipitation of stably transfected HEK293 cells expressing LOX plus ADAMTSL2 or LOX plus ADAMTS10 or parental HEK293 lysate (negative control). LOX migrates as two bands at ~50 kDa. ADAMTSL2 at ~150 kDa, and ADAMTS10 at 150 kDa. Membranes (A,B) were incubated with anti-myc (top) and anti-V5 (bottom). C. Proximity ligation assays performed on HEK293 cells expressing LOX+ADAMTSL2 (I, I′ IV, IV′), LOX+ADAMTS10 (II, II′) and LOX+P85 (III, III′). Anti-LOX and anti-myc antibodies were used to target LOX and ADAMTSL2/ADAMTS10 or P85, respectively. Signal amplification, marked by red signal (top, I-IV) or shown in grayscale (bottom, I′–IV′) is observed only in cells expressing LOX and an ADAMTS protein. In panel IV (control), no primary antibodies we added.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing, Negative Control, Incubation, Ligation, Amplification

    ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.
    Figure Legend Snippet: ADAMTSL2 and ADAMTS10 interact with LOXL2 and LOXL3 A,B. Co-immunoprecipitation from 4 day conditioned medium (A) or the cell lysate (B) from HEK293 cells stably transfected with ADAMTSL2 plus LOXL3 or LOXL3 alone. C . Co-immunoprecipitation of 4 day conditioned medium from stably transfected HEK293 cells expressing either LOXL2 plus ADAMTSL2, LOXL2 plus ADAMTS10, or LOXL2 alone.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Expressing

    LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.
    Figure Legend Snippet: LOX complexes with ADAMTSL4 A . A yeast-two-hybrid (Y2H) screen using LOX as the bait identified three distinct ADAMTSL4 clones (clones 1-3). The selective interaction domain (SID) indicates the consensus interacting region of ADAMTSL4 determined by the overlap of the clones. B . Diploid yeast cells containing both a bait vector (LOX) and prey vector (ADAMTSL4 clone 1) were grown on nutrient permissive or selective plates along with negative controls of empty bait vector, empty prey vector and empty bait and prey vectors. The left-hand panel represents yeast colonies grown on (-)Leu (-)Trp permissive medium to maintain the growth of yeast containing both vectors, while the right-hand panel shows a replicate of the same plate on selective medium. Note that only colonies expressing both LOX and ADAMTSL4 grow on selective medium demonstrating that the two proteins interact. C. Autoradiograph of 35 S-labeled proteins transcribed in the TNT system. Co-immunoprecipitation of lysates expressing Lox plus ADAMTSL4 or Lox plus P85. A band corresponding to Lox is observed slightly under 50 kDa (bottom panel), while that corresponding for ADAMTSL4 is slightly under 150 kDa (top panel). P85 has a molecular mass of 85 kDa. In the TNT reactions where both proteins were translated (as observed in the input lane, right) bands corresponding to both ADAMTSL4 and LOX are observed upon pull down of either one of the proteins. In contrast, P85, which was co-translated with LOX (right input lane), is not observed following LOX pulldown.

    Techniques Used: Clone Assay, Plasmid Preparation, Expressing, Autoradiography, Labeling, Immunoprecipitation

    33) Product Images from "Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors"

    Article Title: Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors

    Journal: eNeuro

    doi: 10.1523/ENEURO.0144-18.2018

    Identification of alternative Gβ 1 γ complexes in rod outer segments of Gγ 1 −/− and WT mice. A–D , Coimmunoprecipitation experiments were performed by incubating Gγ 1 −/− ( A , B ) or WT ( C , D ) rod outer segment lysates with mouse anti-Gγ 2 ( A , C ) or rabbit anti-Gγ 3 ( B , D ) antibodies. Immunoprecipitation with species-matched anti-β-actin (sc-47778) and anti-PSMD1 (ab140682) antibodies were used as negative controls; these antibodies were chosen based on the lack of cross-reactivity with the proteins analyzed in this panel, as evaluated in independent experiments. The data represent one of four similar experiments performed with Gγ 1 −/− or two similar experiments performed with WT outer segment preparations.
    Figure Legend Snippet: Identification of alternative Gβ 1 γ complexes in rod outer segments of Gγ 1 −/− and WT mice. A–D , Coimmunoprecipitation experiments were performed by incubating Gγ 1 −/− ( A , B ) or WT ( C , D ) rod outer segment lysates with mouse anti-Gγ 2 ( A , C ) or rabbit anti-Gγ 3 ( B , D ) antibodies. Immunoprecipitation with species-matched anti-β-actin (sc-47778) and anti-PSMD1 (ab140682) antibodies were used as negative controls; these antibodies were chosen based on the lack of cross-reactivity with the proteins analyzed in this panel, as evaluated in independent experiments. The data represent one of four similar experiments performed with Gγ 1 −/− or two similar experiments performed with WT outer segment preparations.

    Techniques Used: Mouse Assay, Immunoprecipitation

    34) Product Images from "Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD151"

    Article Title: Regulation of Glioblastoma Tumor-Propagating Cells by the Integrin Partner Tetraspanin CD151

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2016.02.003

    CD151:integrin complexes regulate neurosphere cell self-renewal, migration, and Akt activation. (A) GBM1A neurosphere cells collected by cytospin were co-immunostained with antibodies against CD151 and either integrin α3, integrin α6, or integrin β1 (bar = 10μm). CD151 distribution overlaps with the integrins. (B) GBM1A 3F-CD151 cells were treated ± Dox for 48 hours. Brij-O1 collected protein lysates subjected to immunoprecipitation with anti-FLAG specifically precipitated 3F-CD151, integrin α3, integrin α6, and integrin β1 proteins. (C) GBM neurosphere lines GBM1A and GBM1B were infected with lentivirus coding for control shRNA, CD151 shRNA 1, or CD151 shRNA 2. Total cell lysates were extracted and analyzed by immunoblot using antibodies against S 473 phosphorylated Akt (pAkt) and total Akt (tAKT). CD151 inhibition decreased Akt phosphorylation. (D) GBM1A 3F-CD151 and GBM1B 3F-CD151 neurospheres were treated ± Dox. Whole cell extracts were analyzed by immunoblot for pAkt and tAkt. Forced CD151 expression promoted Akt phosphorylation. (E–G) GBM1A and GBM1B cells were treated with anti-CD151 antibody TS151r, which blocks integrin α3 and α6 binding. Isotype IgG was used as control. (E) Equal numbers of viable cells were cultured in 48-well plates with TS151r antibody (or IgG control) for 14 days to form neurospheres. Neurospheres ( > 50 μm diameter) were counted. Neurosphere formation was inhibited by TS151r. (F) Neurosphere cells were plated on laminin-coated Transwell membranes. Migration was evaluated 8 hours later by counting DAPI-stained cells. Cells per field were counted. TS151r inhibited migration on laminin. (G) Whole cell lysates were collected and subjected to immunoblot analysis for pAkt and tAkt. CD151 knockdown inhibits Akt phosphorylation. (H) GBM1A 3F-CD151 cells were treated ± Akt inhibitor MK-2206 (5 μM, Selleckhem, Houston TX) for 1 hour followed by ± Dox for 24 hours to induce 3F-CD151. Neurosphere cells were plated on laminin-coated Transwell membranes. Migration was evaluated 8 hours later by counting DAPI-stained cells. Cells per field were counted. Akt inhibition abrogated CD151-induced cell migration. * P
    Figure Legend Snippet: CD151:integrin complexes regulate neurosphere cell self-renewal, migration, and Akt activation. (A) GBM1A neurosphere cells collected by cytospin were co-immunostained with antibodies against CD151 and either integrin α3, integrin α6, or integrin β1 (bar = 10μm). CD151 distribution overlaps with the integrins. (B) GBM1A 3F-CD151 cells were treated ± Dox for 48 hours. Brij-O1 collected protein lysates subjected to immunoprecipitation with anti-FLAG specifically precipitated 3F-CD151, integrin α3, integrin α6, and integrin β1 proteins. (C) GBM neurosphere lines GBM1A and GBM1B were infected with lentivirus coding for control shRNA, CD151 shRNA 1, or CD151 shRNA 2. Total cell lysates were extracted and analyzed by immunoblot using antibodies against S 473 phosphorylated Akt (pAkt) and total Akt (tAKT). CD151 inhibition decreased Akt phosphorylation. (D) GBM1A 3F-CD151 and GBM1B 3F-CD151 neurospheres were treated ± Dox. Whole cell extracts were analyzed by immunoblot for pAkt and tAkt. Forced CD151 expression promoted Akt phosphorylation. (E–G) GBM1A and GBM1B cells were treated with anti-CD151 antibody TS151r, which blocks integrin α3 and α6 binding. Isotype IgG was used as control. (E) Equal numbers of viable cells were cultured in 48-well plates with TS151r antibody (or IgG control) for 14 days to form neurospheres. Neurospheres ( > 50 μm diameter) were counted. Neurosphere formation was inhibited by TS151r. (F) Neurosphere cells were plated on laminin-coated Transwell membranes. Migration was evaluated 8 hours later by counting DAPI-stained cells. Cells per field were counted. TS151r inhibited migration on laminin. (G) Whole cell lysates were collected and subjected to immunoblot analysis for pAkt and tAkt. CD151 knockdown inhibits Akt phosphorylation. (H) GBM1A 3F-CD151 cells were treated ± Akt inhibitor MK-2206 (5 μM, Selleckhem, Houston TX) for 1 hour followed by ± Dox for 24 hours to induce 3F-CD151. Neurosphere cells were plated on laminin-coated Transwell membranes. Migration was evaluated 8 hours later by counting DAPI-stained cells. Cells per field were counted. Akt inhibition abrogated CD151-induced cell migration. * P

    Techniques Used: Migration, Activation Assay, Immunoprecipitation, Infection, shRNA, Inhibition, Expressing, Binding Assay, Cell Culture, Staining

    35) Product Images from "Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins"

    Article Title: Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201505062

    Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D . (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.
    Figure Legend Snippet: Biochemical evidence for the vacuolar localization of the Dsc complex and a model for the regulation of vacuolar membrane proteins. (A) Subcellular fractionation of WT yeast cells. The whole-cell lysate (T) was separated into P13, P100, and S100 fractions by differential centrifugation and probed with the indicated antibodies. (B) Purified vacuole membrane fraction was compared with the whole-cell lysate (total) by Western blot analysis. Samples that contain an equal amount of Vph1 were loaded in each lane. Approximately 60% of Ubx3 was estimated to localize to the vacuole membrane. (C) Immunoprecipitation experiments from the WT vacuole membrane fraction using preimmune, Dsc2, and Dsc3 antibodies. The immunoprecipitation reaction was probed with the indicated antibodies. (D) Immunoprecipitation experiment from the UBX3-FLAG vacuole membrane fraction using the M2 Flag antibody. The immunoprecipitation reaction was probed with indicated antibodies. (E) Silver staining analysis of the eluates from Fig. 7 D . (F) Two distinct E3 ligase systems converge on the vacuole membrane to regulate different vacuolar membrane transporters via the vReD pathway.

    Techniques Used: Fractionation, Centrifugation, Purification, Western Blot, Immunoprecipitation, Silver Staining

    36) Product Images from "BRCA1 Regulates Acetylation and Ubiquitination of Estrogen Receptor-?"

    Article Title: BRCA1 Regulates Acetylation and Ubiquitination of Estrogen Receptor-?

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2009-0218

    Effect of acetylation-site mutations on BRCA1 regulation of ER-α acetylation. DU-145 cells were pretreated with the indicated siRNA (50 n m ) for 48 h, transfected with wt-ER-α or mutant ER-α, and harvested for immunoprecipitation
    Figure Legend Snippet: Effect of acetylation-site mutations on BRCA1 regulation of ER-α acetylation. DU-145 cells were pretreated with the indicated siRNA (50 n m ) for 48 h, transfected with wt-ER-α or mutant ER-α, and harvested for immunoprecipitation

    Techniques Used: Transfection, Mutagenesis, Immunoprecipitation

    BRCA1 inhibits in vitro acetylation of ER-α by p300. A, In vitro acetylation assays were carried out as described in Materials and Methods . These assays test the ability of p300 (generated from a p300 immunoprecipitation) to acetylate a GST-ER-α
    Figure Legend Snippet: BRCA1 inhibits in vitro acetylation of ER-α by p300. A, In vitro acetylation assays were carried out as described in Materials and Methods . These assays test the ability of p300 (generated from a p300 immunoprecipitation) to acetylate a GST-ER-α

    Techniques Used: In Vitro, Generated, Immunoprecipitation

    BRCA1-I26A mutation does not abrogate BRCA1 association with ER-α in vivo . A, HCC1937 cells, which do not express wtBRCA1, were transfected as indicated and subjected to immunoprecipitation (IP) for ER-α and Western blotting for BRCA1
    Figure Legend Snippet: BRCA1-I26A mutation does not abrogate BRCA1 association with ER-α in vivo . A, HCC1937 cells, which do not express wtBRCA1, were transfected as indicated and subjected to immunoprecipitation (IP) for ER-α and Western blotting for BRCA1

    Techniques Used: Mutagenesis, In Vivo, Transfection, Immunoprecipitation, Western Blot

    37) Product Images from "The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells *"

    Article Title: The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.001685

    siUSP10 redirects CFTR from the recycling pathway to the lysosomal pathway. CFBE cells were transfected with siNeg or siUSP10. Subsequently, co-immunoprecipitation studies were conducted to determine the subcellular location of CFTR in cells treated with
    Figure Legend Snippet: siUSP10 redirects CFTR from the recycling pathway to the lysosomal pathway. CFBE cells were transfected with siNeg or siUSP10. Subsequently, co-immunoprecipitation studies were conducted to determine the subcellular location of CFTR in cells treated with

    Techniques Used: Transfection, Immunoprecipitation

    38) Product Images from "SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination"

    Article Title: SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-019-1069-3

    SKP2 binds to PDCD4 and negatively regulates PDCD4 protein levels. a Lysates from 293 T cells stably transfected with vector or Myc-SKP2 were immunoprecipitated with Myc antibody and subjected to mass spectrometry analysis. PDCD4 was identified as a novel binding partner for SKP2. b , c 293 T cells were harvested for immunoprecipitation with SKP2 antibody (b) or PDCD4 antibody ( c ), followed by immunoblotting. d 35S-labeled in vitro translated SKP2 was used in binding reactions with beads coupled to the PDCD4 peptide K 65 NSSRDSGRGDSVSD 79 or the phosphopeptide K 65 NSpSRDSGRGDSVSD 79 . Bound proteins were eluted and subjected to electrophoresis and autoradiography. e 293 T cells were transfected with indicated plasmids and harvested for immunoprecipitation assay. f SKP2 and PDCD4 expression of SKP2 −/− and WT MEF cells were determined by immunostaining (Scale bars, 10 um). g PDCD4 protein levels were increased in SKP2 −/− MEFs compared with WT MEFs. h PDCD4 and SKP2 protein expression levels in breast cancer cell lines were determined by IB and gray values were analyzed. Spearman rank correlation test was used to test of correlation between the SKP2 and PDCD4 protein expression levels, r = − 1.00, p
    Figure Legend Snippet: SKP2 binds to PDCD4 and negatively regulates PDCD4 protein levels. a Lysates from 293 T cells stably transfected with vector or Myc-SKP2 were immunoprecipitated with Myc antibody and subjected to mass spectrometry analysis. PDCD4 was identified as a novel binding partner for SKP2. b , c 293 T cells were harvested for immunoprecipitation with SKP2 antibody (b) or PDCD4 antibody ( c ), followed by immunoblotting. d 35S-labeled in vitro translated SKP2 was used in binding reactions with beads coupled to the PDCD4 peptide K 65 NSSRDSGRGDSVSD 79 or the phosphopeptide K 65 NSpSRDSGRGDSVSD 79 . Bound proteins were eluted and subjected to electrophoresis and autoradiography. e 293 T cells were transfected with indicated plasmids and harvested for immunoprecipitation assay. f SKP2 and PDCD4 expression of SKP2 −/− and WT MEF cells were determined by immunostaining (Scale bars, 10 um). g PDCD4 protein levels were increased in SKP2 −/− MEFs compared with WT MEFs. h PDCD4 and SKP2 protein expression levels in breast cancer cell lines were determined by IB and gray values were analyzed. Spearman rank correlation test was used to test of correlation between the SKP2 and PDCD4 protein expression levels, r = − 1.00, p

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Binding Assay, Labeling, In Vitro, Electrophoresis, Autoradiography, Expressing, Immunostaining

    39) Product Images from "Aberrant P-cadherin expression is an early event in hyperplastic and dysplastic transformation in the colon"

    Article Title: Aberrant P-cadherin expression is an early event in hyperplastic and dysplastic transformation in the colon

    Journal: Gut

    doi:

    Western blot of HT 29 colorectal cell line (positive control) and colonic adenomas for (A) β-catenin (92 kDa), (B) P-cadherin (120 kDa), and (C) E-cadherin (120 kDa). β-catenin primary immunoprecipitation with subsequent immunoblotting for β-catenin, P-cadherin, and E-cadherin. Both P-cadherin and E-cadherin co-immunoprecipitated with β-catenin in the adenomas analysed.
    Figure Legend Snippet: Western blot of HT 29 colorectal cell line (positive control) and colonic adenomas for (A) β-catenin (92 kDa), (B) P-cadherin (120 kDa), and (C) E-cadherin (120 kDa). β-catenin primary immunoprecipitation with subsequent immunoblotting for β-catenin, P-cadherin, and E-cadherin. Both P-cadherin and E-cadherin co-immunoprecipitated with β-catenin in the adenomas analysed.

    Techniques Used: Western Blot, Positive Control, Immunoprecipitation

    40) Product Images from "Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance"

    Article Title: Mitogen-Activated Protein Kinase Signaling Mediates Opioid-induced Presynaptic NMDA Receptor Activation and Analgesic Tolerance

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14628

    Flowchart diagrams show the timeline of experimental procedures used in the study. Rats were treated with morphine or vehicle for 8 days and used for spinal cord slice recording, behavioral tests (during co-treatment with intrathecal injection of 3 MAPK inhibitors), or co-immunoprecipitation assays. The number of animals used in each group was indicated in parenthesis.
    Figure Legend Snippet: Flowchart diagrams show the timeline of experimental procedures used in the study. Rats were treated with morphine or vehicle for 8 days and used for spinal cord slice recording, behavioral tests (during co-treatment with intrathecal injection of 3 MAPK inhibitors), or co-immunoprecipitation assays. The number of animals used in each group was indicated in parenthesis.

    Techniques Used: Injection, Immunoprecipitation

    Related Articles

    Transfection:

    Article Title: SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson’s disease
    Article Snippet: .. Transfected cells were treated with either diquat (Sigma-Aldrich, UK) dissolved in PBS (phosphate buffered saline; Sigma-Aldrich) or rotenone (Sigma-Aldrich) dissolved in DMSO (dimethyl sulphoxide, Sigma-Aldrich) at a final concentration of 0.2% PBS/DMSO and incubated overnight for 20 h. Cell viability was determined by Alamar Blue reduction assay [ ]. .. Western blotting Following Alamar Blue fluorescence, cell lysates were prepared by scraping the viable cells in native lysis buffer (1% 10× tris buffered saline (TBS), 0.27 M Sucrose, 1% Triton X-100, 1× protease inhibitor cocktail).

    Activation Assay:

    Article Title: MFG-E8-derived Peptide MSP68 is a Cytoskeletal Immunomodulator of Neutrophils that Inhibits Rac1
    Article Snippet: .. BMDNs were pre-treated with either PBS as vehicle or MSP68 at a dose of 10 mM for 30 min, followed by the stimulation with the N-formylmethionine-leucine-phenylalanine ( f -MLP) (Tocris BioSci, Bristol, UK ) at 10 mM for 5 min. After activation with f -MLP, cells were fixed with 3.7% methanol free-formaldehyde in PBS (Sigma, St. Louis, MO) for 10 min at room temperature. .. Slides were then spun at 100 g for 5 min and supernatants were aspirated with fine glass pipette tip.

    Mouse Assay:

    Article Title: 25-Hydroxyvitamin D Depletion Does Not Exacerbate MPTP-Induced Dopamine Neuron Damage in Mice
    Article Snippet: .. Mice then received daily subcutaneous injections of either phosphate buffered saline (PBS) or 15 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP-HCl salt used from Sigma) in PBS for 4 days. ..

    Concentration Assay:

    Article Title: SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson’s disease
    Article Snippet: .. Transfected cells were treated with either diquat (Sigma-Aldrich, UK) dissolved in PBS (phosphate buffered saline; Sigma-Aldrich) or rotenone (Sigma-Aldrich) dissolved in DMSO (dimethyl sulphoxide, Sigma-Aldrich) at a final concentration of 0.2% PBS/DMSO and incubated overnight for 20 h. Cell viability was determined by Alamar Blue reduction assay [ ]. .. Western blotting Following Alamar Blue fluorescence, cell lysates were prepared by scraping the viable cells in native lysis buffer (1% 10× tris buffered saline (TBS), 0.27 M Sucrose, 1% Triton X-100, 1× protease inhibitor cocktail).

    Incubation:

    Article Title: Multiple mechanisms mediate enhanced immunity generated by mAb-inactivated F. tularensis immunogen
    Article Snippet: .. The culture was then spun down at 22,000g for 20 minutes at 4°C, and washed 3 times with PBS, resuspended in 2% paraformaldehyde (Sigma) and incubated 2 hours at room temperature on a rocker. .. Bacteria were then washed 3 more times with PBS and 1×109 organisms were plated on a chocolate agar plate (BD Biosciences) to confirm inactivation.

    Article Title: SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson’s disease
    Article Snippet: .. Transfected cells were treated with either diquat (Sigma-Aldrich, UK) dissolved in PBS (phosphate buffered saline; Sigma-Aldrich) or rotenone (Sigma-Aldrich) dissolved in DMSO (dimethyl sulphoxide, Sigma-Aldrich) at a final concentration of 0.2% PBS/DMSO and incubated overnight for 20 h. Cell viability was determined by Alamar Blue reduction assay [ ]. .. Western blotting Following Alamar Blue fluorescence, cell lysates were prepared by scraping the viable cells in native lysis buffer (1% 10× tris buffered saline (TBS), 0.27 M Sucrose, 1% Triton X-100, 1× protease inhibitor cocktail).

    Western Blot:

    Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
    Article Snippet: .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

    Recombinant:

    Article Title: Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia
    Article Snippet: .. Briefly, microtiter plates (Maxisorp 96well, Nunc #442404) were coated with human recombinant PF4 (10 μg/ml, ProSpec-Tany TechnoGene, Rehovot, Israel) in phosphate buffered saline (PBS) for 2h followed by the addition of either vehicle, heparin (0.002–200 μg/ml, Sigma-Aldrich, Darmstadt, Germany) or ONs (0.001–3 μM). .. After an overnight incubation, the plates were washed 3x with 0.1% Tween-20 and blocked with 3% BSA in PBS for 2 h. After washing with 0.1% Tween-20 plates were incubated with KKO antibody (0.1 μg/ml in 0.005% Tween 20, ThermoFisher Scientific, Waltham, MA, USA) for 1h.

    SDS Page:

    Article Title: Cathepsin L Is Responsible for Processing and Activation of Proheparanase through Multiple Cleavages of a Linker Segment *
    Article Snippet: .. SDS-PAGE and Western Blot Analysis —Cells (1 × 106 ) were lysed in buffer containing 1% Brij 35, 150 m m NaCl, 50 m m Tris-HCl, pH 7.5, or in buffer containing 1% Nonidet P-40, 10 m m EDTA in PBS, both supplemented with a mixture of protease inhibitors (Sigma) ( ). .. Cells were incubated with the lysis buffer for 15–30 min on ice, cell debris were removed by centrifugation and the protein concentration in the supernatants was determined using the Bradford protein assay (Bio-Rad).

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    Millipore co immunoprecipitation co ip buffer
    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using <t>co-immunoprecipitation.</t> β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P
    Co Immunoprecipitation Co Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dip ip buffer
    Correlation between alterations in <t>DNA</t> methylation and gene expression. a and c Heatmaps shows sequence density of significantly-regulated 5hmC regions sorted by RNA-Seq gene expression levels for the 0.1 ( a ) and 0.2 ( c ) Gy dose. Note the cluster of upregulated <t>DIP-Seq</t> data in intragenic regions at the top of the plot. The black bar denotes the position of scaled RefSeq intragenic regions. b and d Gene ontology analyses of the top 500 most up-regulated DIP-Seq regions (by normalized intragenic sequence difference) in the corresponding heatmaps of the 0.1 ( b ) and 0.2 ( d ) Gy doses. e Diagram depicts a subset of genes present in the synapse gene ontology category in D. Note the orthologs and preo/post-synaptic components. f and g UCSC genome browser track depicts DIP-Seq sequence density at two differentially hydroxymethylated gene selected from panels b and d Hatch marks may represent multiple sequences
    Dip Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Journal: BMC Cancer

    Article Title: Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death

    doi: 10.1186/s12885-015-1173-5

    Figure Lengend Snippet: Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Article Snippet: Cells were washed twice with pre-chilled PBS, and then harvested, centrifuged, and suspended in 250 μl Co-Immunoprecipitation (Co-IP) buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.5% NP-40, and 100X protease inhibitor (EMD Millipore).

    Techniques: Modification, Expressing, shRNA, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunofluorescence, Staining, Marker, Fluorescence

    Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

    Journal: Scientific Reports

    Article Title: Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress

    doi: 10.1038/srep16529

    Figure Lengend Snippet: Mechanical stress of ITGB4 phosphorylation in human lung EC. ( A ) Human pulmonary artery EC were grown to confluence on Bioflex plates and then subjected to 18% CS (0–4 h). Cell lysates were then used for immunoprecipitation (IP) using an anti-ITGB4 antibody followed by Western blotting for phosphorylated tyrosine (p-tyrosine; representative blots shown). ( B ) Results of densitometry expressed as p-tyrosine/total ITGB4 are shown (n = 3/condition, *p

    Article Snippet: Immunoprecipitation and Western blotting For immunoprecipitation, cell lysates prepared from EC were incubated in immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2 , 1% Nonidet P-40, 0.4 mM Na3 VO4 , 40 mM NaF, 50 μM okadaic acid, 0.2 mM phenylmethylsulfonyl fluoride, and Calbiochem protease inhibitor mixture III at 1:250 dilution).

    Techniques: Immunoprecipitation, Western Blot

    The LIM protein Ajuba associates with the aPKC-interacting protein p62. (A) Stick figure representation of Ajuba. The gray boxes represent the three LIM domains present in the C-terminal LIM region. The PreLIM region is N terminal. (B) Ajuba and p62 associate in human HepG2 cells. Cell extracts were immunoprecipitated with preimmune serum (PI, lane 1) or Ajuba antiserum (lane 2). Bound products were Western blotted for the presence of p62. In lane 3, 0.5% of the amount of cell extract used for immunoprecipitation (input) was run. (C) Ajuba and p62 colocalize in cells. Fibroblasts were transfected with Flag-tagged p62 alone (panel i), myc-tagged Ajuba alone (panel ii), or both p62-Flagand myc-Ajuba (panels iii to v) and immunostained for p62 (anti-Flag, red, panels i and iii) or Ajuba (anti-myc, green, panels ii and iv). Panel v is a merged image of panels iii and iv. Arrows identify vesicular, endosomal structures. Arrowheads identify focal adhesion sites. (D) Ajuba and LIMD1 but not Zyxin or LPP associate with p62. HEK293 cells were transfected with p62 (Flag-tagged) and myc-tagged LIM proteins, as indicated. p62 was immunoprecipitated (anti-Flag), and bound products were Western blotted for the presence of LIM protein (anti-myc, upper panels) and p62 (anti-Flag, lower panels). For each set, input controls were run (5% of amount used for immunoprecipitation). (E) Mapping of the region of Ajuba that interacts with p62. HEK293 cells were transfected with myc-tagged p62 and Flag-tagged isoforms of Ajuba, as indicated. Ajuba was immunoprecipitated (anti-Flag), and bound products were Western blotted for the presence of p62 (anti-myc, left upper panel) and Ajuba isoforms (anti-Flag, left lower panel). Five percent of the cell extract used for immunoprecipitation was run as an input control (right panels).

    Journal: Molecular and Cellular Biology

    Article Title: The LIM Protein Ajuba Influences Interleukin-1-Induced NF-?B Activation by Affecting the Assembly and Activity of the Protein Kinase C?/p62/TRAF6 Signaling Complex †

    doi: 10.1128/MCB.25.10.4010-4022.2005

    Figure Lengend Snippet: The LIM protein Ajuba associates with the aPKC-interacting protein p62. (A) Stick figure representation of Ajuba. The gray boxes represent the three LIM domains present in the C-terminal LIM region. The PreLIM region is N terminal. (B) Ajuba and p62 associate in human HepG2 cells. Cell extracts were immunoprecipitated with preimmune serum (PI, lane 1) or Ajuba antiserum (lane 2). Bound products were Western blotted for the presence of p62. In lane 3, 0.5% of the amount of cell extract used for immunoprecipitation (input) was run. (C) Ajuba and p62 colocalize in cells. Fibroblasts were transfected with Flag-tagged p62 alone (panel i), myc-tagged Ajuba alone (panel ii), or both p62-Flagand myc-Ajuba (panels iii to v) and immunostained for p62 (anti-Flag, red, panels i and iii) or Ajuba (anti-myc, green, panels ii and iv). Panel v is a merged image of panels iii and iv. Arrows identify vesicular, endosomal structures. Arrowheads identify focal adhesion sites. (D) Ajuba and LIMD1 but not Zyxin or LPP associate with p62. HEK293 cells were transfected with p62 (Flag-tagged) and myc-tagged LIM proteins, as indicated. p62 was immunoprecipitated (anti-Flag), and bound products were Western blotted for the presence of LIM protein (anti-myc, upper panels) and p62 (anti-Flag, lower panels). For each set, input controls were run (5% of amount used for immunoprecipitation). (E) Mapping of the region of Ajuba that interacts with p62. HEK293 cells were transfected with myc-tagged p62 and Flag-tagged isoforms of Ajuba, as indicated. Ajuba was immunoprecipitated (anti-Flag), and bound products were Western blotted for the presence of p62 (anti-myc, left upper panel) and Ajuba isoforms (anti-Flag, left lower panel). Five percent of the cell extract used for immunoprecipitation was run as an input control (right panels).

    Article Snippet: Purified proteins were dialyzed against immunoprecipitation (IP) buffer without NP-40 and concentrated with centrifugal filter units (Millipore).

    Techniques: Immunoprecipitation, Western Blot, Transfection

    Correlation between alterations in DNA methylation and gene expression. a and c Heatmaps shows sequence density of significantly-regulated 5hmC regions sorted by RNA-Seq gene expression levels for the 0.1 ( a ) and 0.2 ( c ) Gy dose. Note the cluster of upregulated DIP-Seq data in intragenic regions at the top of the plot. The black bar denotes the position of scaled RefSeq intragenic regions. b and d Gene ontology analyses of the top 500 most up-regulated DIP-Seq regions (by normalized intragenic sequence difference) in the corresponding heatmaps of the 0.1 ( b ) and 0.2 ( d ) Gy doses. e Diagram depicts a subset of genes present in the synapse gene ontology category in D. Note the orthologs and preo/post-synaptic components. f and g UCSC genome browser track depicts DIP-Seq sequence density at two differentially hydroxymethylated gene selected from panels b and d Hatch marks may represent multiple sequences

    Journal: BMC Genomics

    Article Title: Short- and long-term effects of 56Fe irradiation on cognition and hippocampal DNA methylation and gene expression

    doi: 10.1186/s12864-016-3110-7

    Figure Lengend Snippet: Correlation between alterations in DNA methylation and gene expression. a and c Heatmaps shows sequence density of significantly-regulated 5hmC regions sorted by RNA-Seq gene expression levels for the 0.1 ( a ) and 0.2 ( c ) Gy dose. Note the cluster of upregulated DIP-Seq data in intragenic regions at the top of the plot. The black bar denotes the position of scaled RefSeq intragenic regions. b and d Gene ontology analyses of the top 500 most up-regulated DIP-Seq regions (by normalized intragenic sequence difference) in the corresponding heatmaps of the 0.1 ( b ) and 0.2 ( d ) Gy doses. e Diagram depicts a subset of genes present in the synapse gene ontology category in D. Note the orthologs and preo/post-synaptic components. f and g UCSC genome browser track depicts DIP-Seq sequence density at two differentially hydroxymethylated gene selected from panels b and d Hatch marks may represent multiple sequences

    Article Snippet: The resulting purified DNA was denatured at 95 °C, resuspended in 100 ul of DIP IP buffer, and immunoprecipitated with 1 μg of the highly specific 5-methylcytosine antibody (EMD Millipore) or 2 ul of 5-hydroxymethylcytosine (Active Motif) antibody and Dynal anti-mouse IgG beads.

    Techniques: DNA Methylation Assay, Expressing, Sequencing, RNA Sequencing Assay, DNA Immunoprecipitation Sequencing