immunoprecipitation buffer  (Roche)


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    Structured Review

    Roche immunoprecipitation buffer
    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of <t>immunoprecipitation</t> eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
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    Images

    1) Product Images from "Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity"

    Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2548-14.2015

    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
    Figure Legend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Techniques Used: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

    2) Product Images from "RASSF1C oncogene elicits amoeboid invasion, cancer stemness and invasive EVs via a novel SRC/Rho axis"

    Article Title: RASSF1C oncogene elicits amoeboid invasion, cancer stemness and invasive EVs via a novel SRC/Rho axis

    Journal: bioRxiv

    doi: 10.1101/2021.01.05.425393

    a. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 and MCF7 cell lines transiently transfected either with control or Flag-RASSF1C plasmid. b. Rho-GTP pull-down binding assay in MDA-MB-231 control or RASSF1C over-expressing cells and cotransfected either with SRC plasmids, siRNA against SRC or siNT (non-targeting sequence). Western blot analysis shows Rho activity and pMCLII and pRhoGDI binding. c. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control. d. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA-RASSF1C plasmid, pulled down using a SRC antibody or same species IgG antibody. e. Schematic cartoon recapitulating the proposed RASSF1C/SRC-driven mechanism. f. Top, schematic of RASSF1C domain structure indicating putative mutations in the RA domain affecting RhoA activity. Bottom, immunoprecipitation of MDA-MB-231 transiently transfected with either control (DsRed), DsRASSF1C plasmid, DsRASSF1C-R197W or DsRASSF1C-R199F mutants. Pull-down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies. g. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 cells transiently transfected with RASSF1C or its mutants (R197W, R199F). h. Quantification of amoeboid versus mesenchymal cells in single cell morphology assay in 3D-collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. i. Representative confocal images show mesenchymal or amoeboid cells per field of view of MDA-MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D-collagen and stained with Phalloidin-568 (red) and pMLCII/Alexa 633. Scale bars represent 10 μm. For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
    Figure Legend Snippet: a. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 and MCF7 cell lines transiently transfected either with control or Flag-RASSF1C plasmid. b. Rho-GTP pull-down binding assay in MDA-MB-231 control or RASSF1C over-expressing cells and cotransfected either with SRC plasmids, siRNA against SRC or siNT (non-targeting sequence). Western blot analysis shows Rho activity and pMCLII and pRhoGDI binding. c. Western blot analysis of proteins in MDA-MB-231 cells transiently transfected with siRNA targeting a control sequence (NT) or targeting RASSF1. GAPDH was used as a loading control. d. Immunoprecipitation from MDA-MB-231 transiently transfected with control (pcDNA3) or HA-RASSF1C plasmid, pulled down using a SRC antibody or same species IgG antibody. e. Schematic cartoon recapitulating the proposed RASSF1C/SRC-driven mechanism. f. Top, schematic of RASSF1C domain structure indicating putative mutations in the RA domain affecting RhoA activity. Bottom, immunoprecipitation of MDA-MB-231 transiently transfected with either control (DsRed), DsRASSF1C plasmid, DsRASSF1C-R197W or DsRASSF1C-R199F mutants. Pull-down was performed using a SRC or same species IgG antibody and blotted with indicated antibodies. g. Western blot analysis of Rho-GTP pull-down assay in MDA-MB-231 cells transiently transfected with RASSF1C or its mutants (R197W, R199F). h. Quantification of amoeboid versus mesenchymal cells in single cell morphology assay in 3D-collagen indicating the degree of mesenchymal-amoeboid transition of MDA-MB-231 cells when RASSF1C or the described mutants are expressed. i. Representative confocal images show mesenchymal or amoeboid cells per field of view of MDA-MB-231 cells transfected with DsRed, DsRASSF1C, DsRASSF1C-R197W or DsRASSF1C-R199F, grown in 3D-collagen and stained with Phalloidin-568 (red) and pMLCII/Alexa 633. Scale bars represent 10 μm. For both Rho-GTP pull-downs and immunoprecipitation assays total proteins were used as loading controls. All data are from n=3 independent experiments. Data are represented as mean ± SEM.* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Techniques Used: Western Blot, Pull Down Assay, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Binding Assay, Expressing, Sequencing, Activity Assay, Immunoprecipitation, Morphology Assay, Staining

    3) Product Images from "The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity"

    Article Title: The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

    Journal: bioRxiv

    doi: 10.1101/2020.10.21.336578

    TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (A) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mcherry-TRIM1 transiently transfected into H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (C) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the standard error of the mean. (D) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 days in the presence and absence of 1 μg/ml doxycycline. (E) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments of 150-250 cells each, error bars show standard error of the mean. (F) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments, error bars show standard error of the mean.
    Figure Legend Snippet: TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (A) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mcherry-TRIM1 transiently transfected into H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (C) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the standard error of the mean. (D) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 days in the presence and absence of 1 μg/ml doxycycline. (E) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments of 150-250 cells each, error bars show standard error of the mean. (F) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments, error bars show standard error of the mean.

    Techniques Used: Mutagenesis, Confocal Microscopy, Transfection, Immunoprecipitation, Flow Cytometry, Fluorescence, Plasmid Preparation

    Characterization of LRRK2 co-expressed with 14-3-3 or cytoplasmic TRIM1. (A) Live-cell microscopy in the presence of mCherry-tubulin, GFP-LRRK2, and EBFP-14-3-3 in H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1 C in the presence of HA-ubiquitin in HEK-293T cells. (C) Livecell confocal microscopy of GFP-LRRK2 and mCherry-TRIM1 C transiently transfected into H1299 cells. From left to right, mCherry-TRIM1 C, GFP-LRRK2, merged image. (D) Immunoblot of Rab29 phosphorylation with LRRK2 R1441G in the absence of TRIM1 or with overexpression of Myc-tagged WT TRIM1, TRIM1 C, or TRIM1ΔRF.
    Figure Legend Snippet: Characterization of LRRK2 co-expressed with 14-3-3 or cytoplasmic TRIM1. (A) Live-cell microscopy in the presence of mCherry-tubulin, GFP-LRRK2, and EBFP-14-3-3 in H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1 C in the presence of HA-ubiquitin in HEK-293T cells. (C) Livecell confocal microscopy of GFP-LRRK2 and mCherry-TRIM1 C transiently transfected into H1299 cells. From left to right, mCherry-TRIM1 C, GFP-LRRK2, merged image. (D) Immunoblot of Rab29 phosphorylation with LRRK2 R1441G in the absence of TRIM1 or with overexpression of Myc-tagged WT TRIM1, TRIM1 C, or TRIM1ΔRF.

    Techniques Used: Microscopy, Immunoprecipitation, Confocal Microscopy, Transfection, Over Expression

    Additional characterization of the LRRK2-TRIM1 interaction. Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into (A) A549 cells, (B) SK-N-SH cells, or (C) HEK 293T cells. From left to right, each row shows mCherry-tubulin or mCherry-TRIM1, GFP-LRRK2, merged image. In all lines examined, in the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic, but microtubule localized in the presence of mCherry-TRIM1. (D) Alignment of TRIM18 with TRIM1. Domains labeled above alignment. Red line designates region required for TRIM1 interaction with LRRK2. Double red line designates region of least homology in TRIM1 and TRIM18 dual B-box domain. DualAAA motifs below the sequence designate the mutated amino acids used to make cytoplasmic TRIM1 C variant. (E) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with myc-TRIM18 in HEK293T cells. (F) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with HA-TRIM9 in HEK-293T cells.
    Figure Legend Snippet: Additional characterization of the LRRK2-TRIM1 interaction. Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into (A) A549 cells, (B) SK-N-SH cells, or (C) HEK 293T cells. From left to right, each row shows mCherry-tubulin or mCherry-TRIM1, GFP-LRRK2, merged image. In all lines examined, in the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic, but microtubule localized in the presence of mCherry-TRIM1. (D) Alignment of TRIM18 with TRIM1. Domains labeled above alignment. Red line designates region required for TRIM1 interaction with LRRK2. Double red line designates region of least homology in TRIM1 and TRIM18 dual B-box domain. DualAAA motifs below the sequence designate the mutated amino acids used to make cytoplasmic TRIM1 C variant. (E) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with myc-TRIM18 in HEK293T cells. (F) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with HA-TRIM9 in HEK-293T cells.

    Techniques Used: Confocal Microscopy, Transfection, Labeling, Sequencing, Variant Assay, Immunoprecipitation

    TRIM1 ubiquitinates LRRK2 to regulate its turnover via the proteasome. (A) Co-immunoprecipitation and ubiquitination of FLAG-LRRK2 with myc-TRIM1 or myc-CHIP in the presence of HA-ubiquitin in HEK-293T cells. (B) Schematic of flow cytometric assay using GFP fluorescence to measure GFP-LRRK2 turnover. Doxycycline-inducible GFP-LRRK2 HEK-293T cells are induced for 18 to 24 hours, transfected and doxycycline simultaneously withdrawn, and GFP fluorescence measured after 18-24 hours (additional validation of assay in Figure S2 ). (C) Representative histograms of GFP-LRRK2 fluorescence in the absence or presence of doxycycline followed by TRIM1 or empty vector transfection. (D) Quantification of GFP-LRRK2 levels 24 hours after doxycycline withdrawal in the presence of empty vector (grey bar), TRIM1 (green bar), and ring-finger deleted (ΔRF) TRIM1 (purple bar). (E) Quantification of GFP-LRRK2 levels in the presence of chloroquine at 25 μM for 24 hours (CQ), MG132 at 2 μM for 24 hours, or equivalent volume of DMSO vehicle. (F) Quantification of GFP-LRRK2 fluorescence with TRIM1 knocked down (red bar) compared to cells with non-targeting sgRNA (grey bar) 24 hours after dox withdrawal. (G) Immunoblot of endogenous LRRK2 in lysate of Malme-3M cells with scrambled siRNA (left lane) or endogenous TRIM1 knocked down by targeted siRNA (right lane). (H) Quantification of (G) showing mean value from six independent experiments with error bars showing standard error of the mean. All flow cytometry assays were performed in doxycycline-inducible GFP-LRRK2 HEK-293T cell lines described in Zhao et al . 56 Bar graphs of flow cytometry assays represent normalized median green fluorescence intensity with error bars showing twice the standard error of the mean. All co-immunoprecipitation and flow cytometry assay results show a representative experiment, with the experiment repeated a minimum of 3 times. All histograms or bar charts of flow cytometry results represent at least 10,000 single cells per condition.
    Figure Legend Snippet: TRIM1 ubiquitinates LRRK2 to regulate its turnover via the proteasome. (A) Co-immunoprecipitation and ubiquitination of FLAG-LRRK2 with myc-TRIM1 or myc-CHIP in the presence of HA-ubiquitin in HEK-293T cells. (B) Schematic of flow cytometric assay using GFP fluorescence to measure GFP-LRRK2 turnover. Doxycycline-inducible GFP-LRRK2 HEK-293T cells are induced for 18 to 24 hours, transfected and doxycycline simultaneously withdrawn, and GFP fluorescence measured after 18-24 hours (additional validation of assay in Figure S2 ). (C) Representative histograms of GFP-LRRK2 fluorescence in the absence or presence of doxycycline followed by TRIM1 or empty vector transfection. (D) Quantification of GFP-LRRK2 levels 24 hours after doxycycline withdrawal in the presence of empty vector (grey bar), TRIM1 (green bar), and ring-finger deleted (ΔRF) TRIM1 (purple bar). (E) Quantification of GFP-LRRK2 levels in the presence of chloroquine at 25 μM for 24 hours (CQ), MG132 at 2 μM for 24 hours, or equivalent volume of DMSO vehicle. (F) Quantification of GFP-LRRK2 fluorescence with TRIM1 knocked down (red bar) compared to cells with non-targeting sgRNA (grey bar) 24 hours after dox withdrawal. (G) Immunoblot of endogenous LRRK2 in lysate of Malme-3M cells with scrambled siRNA (left lane) or endogenous TRIM1 knocked down by targeted siRNA (right lane). (H) Quantification of (G) showing mean value from six independent experiments with error bars showing standard error of the mean. All flow cytometry assays were performed in doxycycline-inducible GFP-LRRK2 HEK-293T cell lines described in Zhao et al . 56 Bar graphs of flow cytometry assays represent normalized median green fluorescence intensity with error bars showing twice the standard error of the mean. All co-immunoprecipitation and flow cytometry assay results show a representative experiment, with the experiment repeated a minimum of 3 times. All histograms or bar charts of flow cytometry results represent at least 10,000 single cells per condition.

    Techniques Used: Immunoprecipitation, Chromatin Immunoprecipitation, Flow Cytometry, Fluorescence, Transfection, Plasmid Preparation

    Evaluation of ubiquitination and degradation by TRIM18 and TRIM1ΔRF and verification of flow cytometric system to measure LRRK2 turnover. (A) Co-immunoprecipitation and ubiquitination of FLAG-LRRK2 with myc-TRIM1, myc-TRIM1 ΔRF, or myc-TRIM18 in the presence of HA-ubiquitin in HEK-293T cells. (B) Immunoblot of FLAG-LRRK2 co-transfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection. (C) Quantification of panel (B) with LRRK2 levels normalized to actin. (D) Immunoblot showing LRRK2 levels relative to actin after withdrawal of doxycycline (doxycycline-induced for 18 hours). (E) Histograms of GFP fluorescence from samples immunoblotted in (D). (F) Immunoblot of dox-induced GFP expression co-transfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection.
    Figure Legend Snippet: Evaluation of ubiquitination and degradation by TRIM18 and TRIM1ΔRF and verification of flow cytometric system to measure LRRK2 turnover. (A) Co-immunoprecipitation and ubiquitination of FLAG-LRRK2 with myc-TRIM1, myc-TRIM1 ΔRF, or myc-TRIM18 in the presence of HA-ubiquitin in HEK-293T cells. (B) Immunoblot of FLAG-LRRK2 co-transfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection. (C) Quantification of panel (B) with LRRK2 levels normalized to actin. (D) Immunoblot showing LRRK2 levels relative to actin after withdrawal of doxycycline (doxycycline-induced for 18 hours). (E) Histograms of GFP fluorescence from samples immunoblotted in (D). (F) Immunoblot of dox-induced GFP expression co-transfected with myc-TRIM1 or empty vector control. Time indicates hours after transfection.

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Fluorescence, Expressing

    TRIM1 is a new LRRK2 interacting partner. (A) Diagram of LRRK2 protein domains (ARM: armadillo repeat; ANK: ankyrin repeat; LRR: leucine-rich repeat; ROC: ras of complex proteins; COR: C-terminal of ROC domain). (B) Schematic of LRRK2 interactome in HEK-293T cells. LRRK2 interacting partners are classified radially according to function (aqua: new LRRK2 interacting partners, white: previously identified LRRK2 partners, size of circle indicates fold-change over empty vector control, circles without black outline had no peptides present in empty vector control, arrow indicates TRIM1). FLAG-LRRK2 was immunoprecipitated and interacting partners identified and quantified by MS. Data represent at least four total independent replicates from two experiments and are additionally shown in table S1. (C) Diagram of TRIM1 protein domains (FNIII: fibronectin III domain). (D) Co-immunoprecipitation of myc-TRIM1 with FLAG-LRRK2 in HEK-293T cells. (E) Co-immunoprecipitation of endogenous LRRK2 with TRIM1 in HEK-293T cells. TRIM1 was immunoprecipitated from cells with or without LRRK2 knocked down by siRNA. Asterisk indicates a nonspecific co-reactive band in the whole cell lysate.
    Figure Legend Snippet: TRIM1 is a new LRRK2 interacting partner. (A) Diagram of LRRK2 protein domains (ARM: armadillo repeat; ANK: ankyrin repeat; LRR: leucine-rich repeat; ROC: ras of complex proteins; COR: C-terminal of ROC domain). (B) Schematic of LRRK2 interactome in HEK-293T cells. LRRK2 interacting partners are classified radially according to function (aqua: new LRRK2 interacting partners, white: previously identified LRRK2 partners, size of circle indicates fold-change over empty vector control, circles without black outline had no peptides present in empty vector control, arrow indicates TRIM1). FLAG-LRRK2 was immunoprecipitated and interacting partners identified and quantified by MS. Data represent at least four total independent replicates from two experiments and are additionally shown in table S1. (C) Diagram of TRIM1 protein domains (FNIII: fibronectin III domain). (D) Co-immunoprecipitation of myc-TRIM1 with FLAG-LRRK2 in HEK-293T cells. (E) Co-immunoprecipitation of endogenous LRRK2 with TRIM1 in HEK-293T cells. TRIM1 was immunoprecipitated from cells with or without LRRK2 knocked down by siRNA. Asterisk indicates a nonspecific co-reactive band in the whole cell lysate.

    Techniques Used: Plasmid Preparation, Immunoprecipitation

    LRRK2 ubiquitination by TRIM1 and verification of TRIM1 knockdown. (A) Quantitative MS analysis of LRRK2 K831 ubiquitination in the presence of WT TRIM1, ΔRF TRIM1, or empty vector. (B) All ubiquitin linkages identified by MS analysis of ubiquitinated LRRK2 eluate in the presence of WT TRIM1, ΔRF TRIM1, or empty vector. (C) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 WT or K831R with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (D) Quantification of GFP-LRRK2 levels in the presence of TRIM1, CHIP, or TRIM18. (E) Relative TRIM1 mRNA expression in dox-inducible GFP-LRRK2 HEK-293T cells with dCas9 and either non-targeting sgRNA (grey bar) or four pooled TRIM1-targeting sgRNAs (red bar). (F) Flow cytometric quantification of GFP-LRRK2 levels in TRIM1 knockdown and control lines at 0, 4, 24, and 44 hours after dox withdrawal relative to 0 hours and (G) relative to control cells at all time points. (H) Relative TRIM1 mRNA expression in Malme-3M cells with either scrambled siRNA (grey bar) or TRIM1-targeting siRNA (red bar). (I) Flow cytometric quantification of GFP-LRRK2 levels following LRRK2 inhibitor treatment (100 nM MLi-2 for 24 hours after dox withdrawal), normalized to LRRK2 levels with vehicle treatment. No difference was observed between cells with TRIM1 knocked down (red bars) compared to controls transduced with non-targeting sgRNA (grey bars). All histograms or bar charts of flow cytometry results represent at least 10,000 single cells per condition. Bars show median green fluorescence intensity with error bars showing twice the standard error of the mean.
    Figure Legend Snippet: LRRK2 ubiquitination by TRIM1 and verification of TRIM1 knockdown. (A) Quantitative MS analysis of LRRK2 K831 ubiquitination in the presence of WT TRIM1, ΔRF TRIM1, or empty vector. (B) All ubiquitin linkages identified by MS analysis of ubiquitinated LRRK2 eluate in the presence of WT TRIM1, ΔRF TRIM1, or empty vector. (C) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 WT or K831R with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (D) Quantification of GFP-LRRK2 levels in the presence of TRIM1, CHIP, or TRIM18. (E) Relative TRIM1 mRNA expression in dox-inducible GFP-LRRK2 HEK-293T cells with dCas9 and either non-targeting sgRNA (grey bar) or four pooled TRIM1-targeting sgRNAs (red bar). (F) Flow cytometric quantification of GFP-LRRK2 levels in TRIM1 knockdown and control lines at 0, 4, 24, and 44 hours after dox withdrawal relative to 0 hours and (G) relative to control cells at all time points. (H) Relative TRIM1 mRNA expression in Malme-3M cells with either scrambled siRNA (grey bar) or TRIM1-targeting siRNA (red bar). (I) Flow cytometric quantification of GFP-LRRK2 levels following LRRK2 inhibitor treatment (100 nM MLi-2 for 24 hours after dox withdrawal), normalized to LRRK2 levels with vehicle treatment. No difference was observed between cells with TRIM1 knocked down (red bars) compared to controls transduced with non-targeting sgRNA (grey bars). All histograms or bar charts of flow cytometry results represent at least 10,000 single cells per condition. Bars show median green fluorescence intensity with error bars showing twice the standard error of the mean.

    Techniques Used: Plasmid Preparation, Immunoprecipitation, Chromatin Immunoprecipitation, Expressing, Transduction, Flow Cytometry, Fluorescence

    TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (A) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (ΔBB: TRIM1 construct lacking both B-box domains; ΔCT: TRIM1 lacking C-terminal domain, ΔRF: TRIM1 lacking ring-finger domain; ΔCC: TRIM1 lacking coiled coil domain; ΔFN3: TRIM1 lacking fibronectin III domain; details of constructs in 75 ). (B) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM1 60-715 ; linker denotes TRIM1 60-117 ; BB1 denotes TRIM1 60-164 ; BB1,2 denotes TRIM1 60-212 ). (C) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 822-892 is necessary and sufficient for interaction with TRIM1. (D) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 with alanine mutagenesis within the RL. (E) Live-cell confocal microscopy of GFP-LRRK2 822-982 and mCherry-TRIM1 transiently transfected into H1299 cells. (F) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (LRRK2 822-982 (green)), labelled “RL” and TRIM1 BBOX1 (red). All co-immunoprecipitation experiments are a representative image of at least three independent experiments.
    Figure Legend Snippet: TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (A) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (ΔBB: TRIM1 construct lacking both B-box domains; ΔCT: TRIM1 lacking C-terminal domain, ΔRF: TRIM1 lacking ring-finger domain; ΔCC: TRIM1 lacking coiled coil domain; ΔFN3: TRIM1 lacking fibronectin III domain; details of constructs in 75 ). (B) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM1 60-715 ; linker denotes TRIM1 60-117 ; BB1 denotes TRIM1 60-164 ; BB1,2 denotes TRIM1 60-212 ). (C) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 822-892 is necessary and sufficient for interaction with TRIM1. (D) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 with alanine mutagenesis within the RL. (E) Live-cell confocal microscopy of GFP-LRRK2 822-982 and mCherry-TRIM1 transiently transfected into H1299 cells. (F) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (LRRK2 822-982 (green)), labelled “RL” and TRIM1 BBOX1 (red). All co-immunoprecipitation experiments are a representative image of at least three independent experiments.

    Techniques Used: Immunoprecipitation, Construct, Mutagenesis, Confocal Microscopy, Transfection

    TRIM1 competes with 14-3-3 to bind LRRK2’s regulatory loop and recruit LRRK2 to microtubules. (A) Co-immunoprecipitation of GFP-LRRK2 wild type (WT) and Ser910Ala Ser935Ala (SA) with V5-14-3-3 theta in the presence and absence of myc-TRIM1 in HEK-293T cells. (B) Quantification of (A) showing mean value from three independent experiments with error bars showing the standard error of the mean. (C) Co-immunoprecipitation of GFP-LRRK2 with either V5-14-3-3 theta or myc-TRIM1 in HEK-293T cells. Overlaid immunoblots in color show relative ratio of total to phospho-LRRK2 (total LRRK2 in green, antibody is NeuroMab clone N241A/34; phospho-LRRK2 is in red, antibodies are phospho-Ser910 (Abcam, UDD 1 15(3)) and phospho-Ser935 (Abcam, UDD 2 10(12)). (D) Quantification of (C) showing mean value from three independent experiments with error bars showing the standard error of the mean. (E) Live-cell confocal microscopy of GFP-LRRK2 in the presence of mCherry-TRIM1 and EBPF2-14-3-3 theta. Inset shows GFP-LRRK2 and mCherry-TRIM1 at microtubules with EBFP-14-3-3 theta diffusely cytoplasmic. All live cell imaging and co-immunoprecipitation experiments are a representative image of at least three independent experiments.
    Figure Legend Snippet: TRIM1 competes with 14-3-3 to bind LRRK2’s regulatory loop and recruit LRRK2 to microtubules. (A) Co-immunoprecipitation of GFP-LRRK2 wild type (WT) and Ser910Ala Ser935Ala (SA) with V5-14-3-3 theta in the presence and absence of myc-TRIM1 in HEK-293T cells. (B) Quantification of (A) showing mean value from three independent experiments with error bars showing the standard error of the mean. (C) Co-immunoprecipitation of GFP-LRRK2 with either V5-14-3-3 theta or myc-TRIM1 in HEK-293T cells. Overlaid immunoblots in color show relative ratio of total to phospho-LRRK2 (total LRRK2 in green, antibody is NeuroMab clone N241A/34; phospho-LRRK2 is in red, antibodies are phospho-Ser910 (Abcam, UDD 1 15(3)) and phospho-Ser935 (Abcam, UDD 2 10(12)). (D) Quantification of (C) showing mean value from three independent experiments with error bars showing the standard error of the mean. (E) Live-cell confocal microscopy of GFP-LRRK2 in the presence of mCherry-TRIM1 and EBPF2-14-3-3 theta. Inset shows GFP-LRRK2 and mCherry-TRIM1 at microtubules with EBFP-14-3-3 theta diffusely cytoplasmic. All live cell imaging and co-immunoprecipitation experiments are a representative image of at least three independent experiments.

    Techniques Used: Immunoprecipitation, Western Blot, Confocal Microscopy, Live Cell Imaging

    4) Product Images from "Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion"

    Article Title: Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007471

    HEV ORF3 protein oligomerizes in mammalian cells. ( A ) Sequence analysis of ORF3 protein. Amino acid sequences of ORF3 from HEV genotypes 1–8 (GenBank accession numbers AB740232, AF444002, M74506, AJ272108, AB573435, AB856243, KJ496144 and KX387866) were aligned using ClustalW [ 52 ]. Segment aa 30–53 predicted as transmembrane passage by TMPred is boxed in grey [ 53 ]. A consensus secondary structure was predicted using algorithms MLRC, DSC and PHD (available at https://npsa-prabi.ibcp.fr ) and is shown below the alignment (c, random coil; h, α-helix; e, extended strand;?, discrepant prediction). The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [ 52 ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P
    Figure Legend Snippet: HEV ORF3 protein oligomerizes in mammalian cells. ( A ) Sequence analysis of ORF3 protein. Amino acid sequences of ORF3 from HEV genotypes 1–8 (GenBank accession numbers AB740232, AF444002, M74506, AJ272108, AB573435, AB856243, KJ496144 and KX387866) were aligned using ClustalW [ 52 ]. Segment aa 30–53 predicted as transmembrane passage by TMPred is boxed in grey [ 53 ]. A consensus secondary structure was predicted using algorithms MLRC, DSC and PHD (available at https://npsa-prabi.ibcp.fr ) and is shown below the alignment (c, random coil; h, α-helix; e, extended strand;?, discrepant prediction). The degree of aa physicochemical conservation at each position is shown on the bottom line and can be inferred with the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [ 52 ]. ( B ) ORF3 protein oligomerization was analyzed by FLAG immunoprecipitation. Lysates (Input) of U-2 OS cells transfected with pCMVORF3-HA and/or pCMVORF3-FLAG as well as immunoprecipitates (IP: FLAG) were subjected to immunoblot with either specific anti-FLAG or anti-HA antibodies. The presence of a strong signal for ORF3-HA after pull-down of ORF3-FLAG indicates oligomerization of ORF3 protein in cells. ( C ) FRET analyses reveal oligomerization of HEV ORF3. CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein) fused to the C-termini of HEV ORF3 segments aa 1–113, 1–94, 1–70, 1–53, 28–113 or 53–113 were co-expressed in U-2 OS cells. FRET analyses were performed by the acceptor photobleaching method as described in the Materials and Methods section. The CFP-YFP fusion protein and cotransfection of unfused CFP and YFP served as positive and negative controls, respectively. Box-and-whisker plots represent the median FRET efficiency (FRETeff) values of 20 measurements (middle line), the values from the lower to the upper quartile (central box), and the minimum and maximum values (vertical line). The significance of the observed differences was assessed as described in Materials and Methods (*, P

    Techniques Used: Sequencing, Immunoprecipitation, Transfection, Cotransfection, Whisker Assay

    HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).
    Figure Legend Snippet: HEV ORF3 protein is palmitoylated. Protein lysates from S10-3 cells transfected with pCMVORF3, pCMVORF3 C1-4 , pCMVORF3 C45-8 or pCMVORF3_gt1 and from Hep293TT cells replicating the full-length p6 or 83–2 HEV clone were prepared 1 or 6 days post-transfection, respectively, and subjected to immunoprecipitation with either anti-ORF3 pAb (+) or non-relevant rabbit serum (-). After immunoprecipitation, the elution samples were separated by 17% SDS-PAGE and subjected to either immunoblot with anti-ORF3 pAb followed by chemiluminescence revelation or autoradiography (40 d of exposure).

    Techniques Used: Transfection, Immunoprecipitation, SDS Page, Autoradiography

    5) Product Images from "BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity"

    Article Title: BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016828

    BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).
    Figure Legend Snippet: BAG3 recognizes folding status of αB-crystallin. (A) BAG3 binds to mutant type of αB-crystallin strongly. Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). (B) Direct recognition of mutant αB-crystallin by BAG3. Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. Mutated αB-crystallin preferentially binds to BAG3. Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).

    Techniques Used: Mutagenesis, SDS Page, Immunoprecipitation, Expressing, Purification, Incubation, Pull Down Assay, Staining

    6) Product Images from "Phosphorylation of kinesin light chain 1 at serine 460 modulates binding and trafficking of calsyntenin-1"

    Article Title: Phosphorylation of kinesin light chain 1 at serine 460 modulates binding and trafficking of calsyntenin-1

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.075168

    KLC1ser460 is phosphorylated by ERK and inhibition of ERK increases binding of KLC1 to calsyntenin-1. ( A ) Inhibition of ERK promotes binding of calsyntenin-1 to KLC1wt but has no effect on its binding to KLC1ser460ala in immunoprecipitation (IP) assays
    Figure Legend Snippet: KLC1ser460 is phosphorylated by ERK and inhibition of ERK increases binding of KLC1 to calsyntenin-1. ( A ) Inhibition of ERK promotes binding of calsyntenin-1 to KLC1wt but has no effect on its binding to KLC1ser460ala in immunoprecipitation (IP) assays

    Techniques Used: Inhibition, Binding Assay, Immunoprecipitation

    7) Product Images from "The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing"

    Article Title: The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

    Journal: Genes & Development

    doi: 10.1101/gad.254631.114

    G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1
    Figure Legend Snippet: G4 antigenicity of piRNA precursor transcripts increases in Mov10l1 mutant mice. ( A ) RNA immunoprecipitation of piRNA precursor transcripts using the BG4 antibody that specifically recognizes RNA G4s using testis lysates from wild-type (WT) and Mov10l1

    Techniques Used: Mutagenesis, Mouse Assay, Immunoprecipitation

    8) Product Images from "Mitochondrial energy dysfunction induces remodeling of the cardiac mitochondrial protein acylome"

    Article Title: Mitochondrial energy dysfunction induces remodeling of the cardiac mitochondrial protein acylome

    Journal: bioRxiv

    doi: 10.1101/2021.01.31.429057

    Site-specific expression and activity of IDH2 acetylation and malonylation mimics. (A) IDH2 lysine residues hyperacetylated (Kac) and hypermalonylated (Kmal) in response to SLC25A3 deletion. Residues modified by both PTMs in bold. (B) Structure of the IDH2 dimer bound to isocitrate. Lysine resides acetylated (red), malonylated (yellow), or targeted by both PTMs (purple) in response to SLC25A3 deletion are indicated. (C) IDH2 was immunoprecipitated from protein lysates from Slc25a3 fl/fl or Slc25a3 fl/flxMCM hearts and probed by immunoblotting by anti-Kac or anti-Kmal antibodies to confirm enhanced acetylation/malonylation of IDH2 in response to SLC25A3 deletion. Immunoblotting with anti-IDH2 antibodies was conducted to validate pull down of IDH2. Immunoprecipitation was also conducted with IgG antibodies as a control. (D) IDH2 activity assay performed on cardiac mitochondria isolated from Slc25a3 fl/fl and Slc25a3 fl/flxMCM animals 10 weeks post-tamoxifen administration. (E) Representation of K180 (purple) and isocitrate in the IDH2 binding pocket. (F) Western blot analyses of IDH2 expression in WT and IDH2 KO clones. Α-tubulin was used as a protein loading control. (G) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the pcDNA3.1 vector alone (empty), WT IDH2, IDH2 K180E, or IDH2 K180Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. (H) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the empty vector, WT IDH2, IDH2 K48E, or IDH2 K48Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. (I) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the empty vector, WT IDH2, IDH2 K263E, or IDH2 K263Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. For all graphs, values presented as mean ± SEM. For (D-F), one way ANOVA followed by Welch’s test was used for statistical analysis with P
    Figure Legend Snippet: Site-specific expression and activity of IDH2 acetylation and malonylation mimics. (A) IDH2 lysine residues hyperacetylated (Kac) and hypermalonylated (Kmal) in response to SLC25A3 deletion. Residues modified by both PTMs in bold. (B) Structure of the IDH2 dimer bound to isocitrate. Lysine resides acetylated (red), malonylated (yellow), or targeted by both PTMs (purple) in response to SLC25A3 deletion are indicated. (C) IDH2 was immunoprecipitated from protein lysates from Slc25a3 fl/fl or Slc25a3 fl/flxMCM hearts and probed by immunoblotting by anti-Kac or anti-Kmal antibodies to confirm enhanced acetylation/malonylation of IDH2 in response to SLC25A3 deletion. Immunoblotting with anti-IDH2 antibodies was conducted to validate pull down of IDH2. Immunoprecipitation was also conducted with IgG antibodies as a control. (D) IDH2 activity assay performed on cardiac mitochondria isolated from Slc25a3 fl/fl and Slc25a3 fl/flxMCM animals 10 weeks post-tamoxifen administration. (E) Representation of K180 (purple) and isocitrate in the IDH2 binding pocket. (F) Western blot analyses of IDH2 expression in WT and IDH2 KO clones. Α-tubulin was used as a protein loading control. (G) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the pcDNA3.1 vector alone (empty), WT IDH2, IDH2 K180E, or IDH2 K180Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. (H) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the empty vector, WT IDH2, IDH2 K48E, or IDH2 K48Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. (I) Western blot analyses of IDH2 expression in IDH2 KO cells re-expressing either the empty vector, WT IDH2, IDH2 K263E, or IDH2 K263Q constructs, and the IDH2 activity assays on mitochondria isolated from these cell lines. For all graphs, values presented as mean ± SEM. For (D-F), one way ANOVA followed by Welch’s test was used for statistical analysis with P

    Techniques Used: Expressing, Activity Assay, Modification, Immunoprecipitation, Isolation, Binding Assay, Western Blot, Clone Assay, Plasmid Preparation, Construct

    Acetylation of SIRT5 modulates malonylation status of mitochondrial proteins. (A) Cardiac malonyl-CoA levels in Slc25a3 fl/fl versus Slc25a3 fl/flxMCM mice at 10 weeks post-tamoxifen administration. (B) Western blot analysis of SIRT5 expression in total heart protein lysates from Slc25a3 fl/fl versus Slc25a3 fl/flxMCM animals. GAPDH was used as a loading control. (C) Immunoprecipitation confirmation of enhanced SIRT5 acetylation in Slc25a3 fl/flxMCM versus Slc25a3 fl/fl hearts. Acetylated proteins were immunoprecipated with anti-Kac antibodies followed by immunoblotting with an antibody against SIRT5. An anti-HA antibody was used as an immunoprecipitation control. (D) Western blot analysis of SIRT5 expression in HEK293 WT versus SIRT5 KO clones. α-tubulin was used as a protein loading control. (E) Western blot analysis of SIRT5 expression in SIRT5 KO cells re-expressing a pcDNA3.1 empty vector, WT SIRT5 or, the SIRT5 K203Q mutant. GAPDH was used as a loading control. (F) Representative western blot of protein lysine malonylation (Kmal) and succinylation (Ksuc) of mitochondria isolated from SIRT5 KO cells, or SIRT5 KO cells re-expressing the empty vector, WT SIRT5, or SIRT5 K203Q. VDAC was used as a loading control. (G) Quantification of protein malonylation in SIRT5 KO cells re-expressing the WT and K203Q SIRT5 constructs. (H) Quantification of protein succinylation in SIRT5 KO cells re-expressing the WT and K203Q SIRT5 constructs. One way ANOVA followed by Welch’s test was used for statistical analysis with P
    Figure Legend Snippet: Acetylation of SIRT5 modulates malonylation status of mitochondrial proteins. (A) Cardiac malonyl-CoA levels in Slc25a3 fl/fl versus Slc25a3 fl/flxMCM mice at 10 weeks post-tamoxifen administration. (B) Western blot analysis of SIRT5 expression in total heart protein lysates from Slc25a3 fl/fl versus Slc25a3 fl/flxMCM animals. GAPDH was used as a loading control. (C) Immunoprecipitation confirmation of enhanced SIRT5 acetylation in Slc25a3 fl/flxMCM versus Slc25a3 fl/fl hearts. Acetylated proteins were immunoprecipated with anti-Kac antibodies followed by immunoblotting with an antibody against SIRT5. An anti-HA antibody was used as an immunoprecipitation control. (D) Western blot analysis of SIRT5 expression in HEK293 WT versus SIRT5 KO clones. α-tubulin was used as a protein loading control. (E) Western blot analysis of SIRT5 expression in SIRT5 KO cells re-expressing a pcDNA3.1 empty vector, WT SIRT5 or, the SIRT5 K203Q mutant. GAPDH was used as a loading control. (F) Representative western blot of protein lysine malonylation (Kmal) and succinylation (Ksuc) of mitochondria isolated from SIRT5 KO cells, or SIRT5 KO cells re-expressing the empty vector, WT SIRT5, or SIRT5 K203Q. VDAC was used as a loading control. (G) Quantification of protein malonylation in SIRT5 KO cells re-expressing the WT and K203Q SIRT5 constructs. (H) Quantification of protein succinylation in SIRT5 KO cells re-expressing the WT and K203Q SIRT5 constructs. One way ANOVA followed by Welch’s test was used for statistical analysis with P

    Techniques Used: Mouse Assay, Western Blot, Expressing, Immunoprecipitation, Clone Assay, Plasmid Preparation, Mutagenesis, Isolation, Construct

    9) Product Images from "Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma"

    Article Title: Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma

    Journal: Oncogenesis

    doi: 10.1038/s41389-018-0050-x

    TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Binding Assay, Immunoprecipitation, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

    10) Product Images from "NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics"

    Article Title: NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4604-11.2012

    BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.
    Figure Legend Snippet: BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.

    Techniques Used: Western Blot, Immunoprecipitation, Transfection, Recombinant, Confocal Microscopy

    11) Product Images from "Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation"

    Article Title: Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.156778

    Epfn promotes HaCaT cell differentiation through regulating Notch1. (A) Expression of keratinocyte marker genes as revealed by qPCR in HaCaT cells transfected with either Mock or Epfn expression vectors. (B) Co-immunoprecipitation assay of Epfn and E2F1 in HaCaT cells transfected with E2F1 (HA tag) and Epfn (VSV tag) expression vectors. IP, immunoprecipitated; IB, immunoblotted. (C) E2F activity assay in HaCaT cells transfected with Mock and Epfn expression vectors. (D) RBP-Jk reporter activity in HaCaT cells transfected with either Mock or Epfn expression vectors. Quantitative data in A,C,D show the mean+s.e.m. (three independent experiments); ** P
    Figure Legend Snippet: Epfn promotes HaCaT cell differentiation through regulating Notch1. (A) Expression of keratinocyte marker genes as revealed by qPCR in HaCaT cells transfected with either Mock or Epfn expression vectors. (B) Co-immunoprecipitation assay of Epfn and E2F1 in HaCaT cells transfected with E2F1 (HA tag) and Epfn (VSV tag) expression vectors. IP, immunoprecipitated; IB, immunoblotted. (C) E2F activity assay in HaCaT cells transfected with Mock and Epfn expression vectors. (D) RBP-Jk reporter activity in HaCaT cells transfected with either Mock or Epfn expression vectors. Quantitative data in A,C,D show the mean+s.e.m. (three independent experiments); ** P

    Techniques Used: Cell Differentiation, Expressing, Marker, Real-time Polymerase Chain Reaction, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Activity Assay

    12) Product Images from "A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells"

    Article Title: A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200804154

    CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.
    Figure Legend Snippet: CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.

    Techniques Used: Pull Down Assay, Expressing, Generated, SPR Assay, Flow Cytometry, Binding Assay, Immunoprecipitation

    13) Product Images from "Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading"

    Article Title: Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22503-7

    Specific effects of alg-2 on the neuronal “translatome”. a Schematic representation of cell-type-specific polysome immunoprecipitation setup. Single copy FLAG-tagged RPL-18 protein driven by the neuron-specific rgef-1 promoter allows purification of neuron-specific translating mRNAs from either wild-type or alg-2 mutant backgrounds. Scale bars, 50 µm. b Scatterplot showing expression levels (log 2 CPM, counts per million) of mRNAs immunoprecipitated with polysomes from neurons in alg-2 mutant compared to wild-type worms. Red points represent significantly differentially upregulated or downregulated genes with a FDR value of
    Figure Legend Snippet: Specific effects of alg-2 on the neuronal “translatome”. a Schematic representation of cell-type-specific polysome immunoprecipitation setup. Single copy FLAG-tagged RPL-18 protein driven by the neuron-specific rgef-1 promoter allows purification of neuron-specific translating mRNAs from either wild-type or alg-2 mutant backgrounds. Scale bars, 50 µm. b Scatterplot showing expression levels (log 2 CPM, counts per million) of mRNAs immunoprecipitated with polysomes from neurons in alg-2 mutant compared to wild-type worms. Red points represent significantly differentially upregulated or downregulated genes with a FDR value of

    Techniques Used: Immunoprecipitation, Purification, Mutagenesis, Expressing

    Cell-type-specific profiling of AGO-loaded miRNAs. a Overview of cell-type-specific miRNA profiling technique. HA-tagged copies of ALG-1 or ALG-2 were driven by cell-type-specific promoters, allowing immunoprecipitation of AGO-loaded miRNAs from individual tissue types from total worm homogenates. b Whole animal fluorescence images of cell-type-specific HA ALG-1 and HA ALG-2 lines demonstrating specific expression from intestine, body wall muscles, or neurons. Scale bars, 50 µm. Asterisks indicate intestine autofluorescence. Images are representative of > 50 independent animals. c Western blot analysis of input (left) and immunoprecipitated (right) AGO complexes. All images are from the same blot and are equally exposed. Coomassie blue serves as a loading control. d RNA gel blot analysis of input (left) and AGO-immunoprecipitated miRNAs (right). U6 serves as a loading control. e Quantitative RT-PCR on immunoprecipitated AGO:miRNA complexes showing either enrichment > 1 or lack of enrichment
    Figure Legend Snippet: Cell-type-specific profiling of AGO-loaded miRNAs. a Overview of cell-type-specific miRNA profiling technique. HA-tagged copies of ALG-1 or ALG-2 were driven by cell-type-specific promoters, allowing immunoprecipitation of AGO-loaded miRNAs from individual tissue types from total worm homogenates. b Whole animal fluorescence images of cell-type-specific HA ALG-1 and HA ALG-2 lines demonstrating specific expression from intestine, body wall muscles, or neurons. Scale bars, 50 µm. Asterisks indicate intestine autofluorescence. Images are representative of > 50 independent animals. c Western blot analysis of input (left) and immunoprecipitated (right) AGO complexes. All images are from the same blot and are equally exposed. Coomassie blue serves as a loading control. d RNA gel blot analysis of input (left) and AGO-immunoprecipitated miRNAs (right). U6 serves as a loading control. e Quantitative RT-PCR on immunoprecipitated AGO:miRNA complexes showing either enrichment > 1 or lack of enrichment

    Techniques Used: Immunoprecipitation, Fluorescence, Expressing, Western Blot, Quantitative RT-PCR

    14) Product Images from "Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88"

    Article Title: Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1402598

    Co-immunoprecipitation of TLR-4, FAK and MyD88 using Dynabeads Protein G. After LPS treatment (5 d), Caco-2 monolayers were lysed, and immunoprecipitated with TLR-4 antibody, then phospho-FAK, FAK and MyD88 were detected by Western blot analysis. n=4. Densitometry analysis showed a significant increase in phospho-FAK compared to control and no change in total FAK and MyD88 expression. *, p
    Figure Legend Snippet: Co-immunoprecipitation of TLR-4, FAK and MyD88 using Dynabeads Protein G. After LPS treatment (5 d), Caco-2 monolayers were lysed, and immunoprecipitated with TLR-4 antibody, then phospho-FAK, FAK and MyD88 were detected by Western blot analysis. n=4. Densitometry analysis showed a significant increase in phospho-FAK compared to control and no change in total FAK and MyD88 expression. *, p

    Techniques Used: Immunoprecipitation, Western Blot, Expressing

    15) Product Images from "Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus"

    Article Title: Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus

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

    doi: 10.1073/pnas.0708368104

    Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.
    Figure Legend Snippet: Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.

    Techniques Used: Transgenic Assay, Immunoprecipitation, Mutagenesis

    16) Product Images from "Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115"

    Article Title: Phospholipase C γ1 regulates early secretory trafficking and cell migration via interaction with p115

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-03-0178

    (A) Coimmunoprecipitation of PLCG1 (IP: PLCG1) and p115 (IP: p115) from HeLa cell lysate. Input, the input material of the immunoprecipitation (5%). Neg. Control, the negative control in which the immunoprecipitation was performed in the absence of antibody but in the presence of protein G–Sepharose beads. Immunoprecipitated PLCG1 or p115 was eluted and immunoblotted against p115 and PLCG1, respectively (upper gel). Blots were stripped and immunoblotted against PLCG1 and p115 (lower gel). (B) Schematic depiction of the domains in PLCG1. (C) HeLa cells were transfected with a plasmid encoding wild-type GFP-tagged PLCG1 (PLCG1-WT) or truncation mutants lacking 19, 39, 60, and 81 C-terminal amino acids (PLCG1-Δ19, -Δ39, -Δ60, and -Δ81, respectively). In addition, a truncation mutant lacking the C2 domain was also used (PLCG1-ΔC2). After 24 h, cells were lysed and the lysate subjected to immunoprecipitation with GFP-tap beads (ChromoTek). The immunoprecipitated material was subjected to SDS–PAGE and immunoblotted against p115. The blot was stripped and probed with an antibody against GFP (to detect PLCG1). (D) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding either GFP or GFP-tagged PLCG1-ΔC2. After 8 h, cells were plated into ibidi migration inserts. Cell migration was initiated by removing the insert, and cells were allowed to migrate for 18 h. (E, F) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding GFP or GFP-tagged PLCG1-ΔC2 (E) or PLCG1-Δ81 (F). Asterisks indicate statistically significant differences from control (* p
    Figure Legend Snippet: (A) Coimmunoprecipitation of PLCG1 (IP: PLCG1) and p115 (IP: p115) from HeLa cell lysate. Input, the input material of the immunoprecipitation (5%). Neg. Control, the negative control in which the immunoprecipitation was performed in the absence of antibody but in the presence of protein G–Sepharose beads. Immunoprecipitated PLCG1 or p115 was eluted and immunoblotted against p115 and PLCG1, respectively (upper gel). Blots were stripped and immunoblotted against PLCG1 and p115 (lower gel). (B) Schematic depiction of the domains in PLCG1. (C) HeLa cells were transfected with a plasmid encoding wild-type GFP-tagged PLCG1 (PLCG1-WT) or truncation mutants lacking 19, 39, 60, and 81 C-terminal amino acids (PLCG1-Δ19, -Δ39, -Δ60, and -Δ81, respectively). In addition, a truncation mutant lacking the C2 domain was also used (PLCG1-ΔC2). After 24 h, cells were lysed and the lysate subjected to immunoprecipitation with GFP-tap beads (ChromoTek). The immunoprecipitated material was subjected to SDS–PAGE and immunoblotted against p115. The blot was stripped and probed with an antibody against GFP (to detect PLCG1). (D) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding either GFP or GFP-tagged PLCG1-ΔC2. After 8 h, cells were plated into ibidi migration inserts. Cell migration was initiated by removing the insert, and cells were allowed to migrate for 18 h. (E, F) HeLa cells were transfected with the indicated siRNAs. After 48 h, cells were transfected with plasmids encoding GFP or GFP-tagged PLCG1-ΔC2 (E) or PLCG1-Δ81 (F). Asterisks indicate statistically significant differences from control (* p

    Techniques Used: Immunoprecipitation, Negative Control, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Migration

    17) Product Images from "Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93"

    Article Title: Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93

    Journal: Journal of Virology

    doi: 10.1128/JVI.00351-16

    Interaction of pUL77 with pUL93. (A) RPE-1 cells infected with HCMV AD169 were transfected at 48 h p.i. with pGEX_UL93 or pGEX5x-1. Cells were lysed at 24 h posttransfection, and immunoprecipitation (IP) was performed with antibodies to pUL77. Cell extracts and coimmunoprecipitated (Co-IP) proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. (B) HEK293T cells were cotransfected with pGEX_UL93 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to GST. Cell extracts and coimmunoprecipitated proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. Molecular mass markers (M) are indicated on the left of each panel.
    Figure Legend Snippet: Interaction of pUL77 with pUL93. (A) RPE-1 cells infected with HCMV AD169 were transfected at 48 h p.i. with pGEX_UL93 or pGEX5x-1. Cells were lysed at 24 h posttransfection, and immunoprecipitation (IP) was performed with antibodies to pUL77. Cell extracts and coimmunoprecipitated (Co-IP) proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. (B) HEK293T cells were cotransfected with pGEX_UL93 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to GST. Cell extracts and coimmunoprecipitated proteins were subjected to SDS-PAGE prior to Western blot analyses with antibodies to pUL77 and GST. Molecular mass markers (M) are indicated on the left of each panel.

    Techniques Used: Infection, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Mutagenesis

    Interaction of pUL77 with terminase subunits pUL56 and pUL89. (A) HFFs were infected with HCMV AD169 prior to coimmunoprecipitation (Co-IP) with an antibody specific to pUL77. Precipitated proteins were subjected to Western blot analyses with antibodies to pUL77, IE1, pUL56, and pUL89. (B) HEK293T cells were cotransfected with pEYFP_UL56 and pcDNA_UL77 or two mutant variants of pcDNA_UL77. Immunoprecipitation (IP) was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GFP. (C) HEK293T cells were cotransfected with pGEX_UL89 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GST and the His tag. Molecular mass markers (M) are indicated on the left.
    Figure Legend Snippet: Interaction of pUL77 with terminase subunits pUL56 and pUL89. (A) HFFs were infected with HCMV AD169 prior to coimmunoprecipitation (Co-IP) with an antibody specific to pUL77. Precipitated proteins were subjected to Western blot analyses with antibodies to pUL77, IE1, pUL56, and pUL89. (B) HEK293T cells were cotransfected with pEYFP_UL56 and pcDNA_UL77 or two mutant variants of pcDNA_UL77. Immunoprecipitation (IP) was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GFP. (C) HEK293T cells were cotransfected with pGEX_UL89 and pcDNA_UL77 or mutant variants of pcDNA_UL77. Immunoprecipitation was performed with antibodies to the His tag. Cell extracts were subjected to Western blot analyses with antibodies to GST and the His tag. Molecular mass markers (M) are indicated on the left.

    Techniques Used: Infection, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Immunoprecipitation

    18) Product Images from "Negative Regulation of the RalGAP Complex by 14-3-3 *"

    Article Title: Negative Regulation of the RalGAP Complex by 14-3-3 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.426106

    RGC2 T715 phosphorylation is required for 14-3-3 binding. A , schematic of λ-phosphatase protection assay. IP , immunoprecipitation. B , λ-phosphatase protection assay. 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2.
    Figure Legend Snippet: RGC2 T715 phosphorylation is required for 14-3-3 binding. A , schematic of λ-phosphatase protection assay. IP , immunoprecipitation. B , λ-phosphatase protection assay. 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2.

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation

    14-3-3 binding does not affect RalGAP complex in vitro catalytic activity. A , 293A cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using
    Figure Legend Snippet: 14-3-3 binding does not affect RalGAP complex in vitro catalytic activity. A , 293A cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using

    Techniques Used: Binding Assay, In Vitro, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

    14-3-3 interacts with the RalGAP complex. A , 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using anti-FLAG antibody ( left panel
    Figure Legend Snippet: 14-3-3 interacts with the RalGAP complex. A , 293T cells were transfected with empty vector or HA-RGC1 and FLAG-RGC2 as indicated. Two days after transfection, cell lysates were subjected to immunoprecipitation ( IP ) using anti-FLAG antibody ( left panel

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation

    19) Product Images from "NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics"

    Article Title: NP1 Regulates Neuronal Activity-Dependent Accumulation of BAX in Mitochondria and Mitochondrial Dynamics

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4604-11.2012

    BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.
    Figure Legend Snippet: BAX immunoprecipitates NP1 and NP1 colocalizes with active BAX in mitochondria. A–C , Representative Western blots of total protein lysate (TL), the eluate of BAX immunoprecipitation (IP; Bax), and the eluate of IgG immunoprecipitation controls (IgG). A , Immunoprecipitation of BAX from total brain lysates. Rat brains were homogenized, and protein lysates prepared as described in Materials and Methods. B , Immunoprecipitation of BAX from CGN lysates. CGNs were treated with low K + for 2 h. C , Immunoprecipitation of BAX from HEK 293T cell lysates transfected with recombinant rat Bax and NP1 cDNAs. D , Confocal microscopy studies were performed in mature CGNs in culture after 4 h of K + deprivation. Orthogonal sectioning shows colocalization between NP1 and active BAX in mitochondria. Colocalization between NP1 and MtDsRed is seen as yellow, between NP1 and BAX as cyan, and between BAX and MtDsRed as magenta, and colocalization between all three proteins is shown in white. Scale bar, 5 μm.

    Techniques Used: Western Blot, Immunoprecipitation, Transfection, Recombinant, Confocal Microscopy

    20) Product Images from "The Arkadia-ESRP2 axis suppresses tumor progression: analyses in clear-cell renal cell carcinoma"

    Article Title: The Arkadia-ESRP2 axis suppresses tumor progression: analyses in clear-cell renal cell carcinoma

    Journal: Oncogene

    doi: 10.1038/onc.2015.412

    Modulation of the splicing function of ESRP2 by Arkadia through ubiquitination. ( a and b ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( a ) or ESRP2 ( b ), Arkadia wild-type or CA mutant, and ubiquitin. IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin; WT, wild-type. ( c and d ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( c ) or ESRP2 ( d ), WT Arkadia, and ubiquitin WT or its mutants. 7KR: all seven lysine residues are substituted by arginine residues; 6, 11, 27, 29, 33, 48 and 63K: one lysine residue is intact, but the others are substituted by arginine residues. KR, lysine-to-arginine mutation. ( e ) Schematic representation of ESRP2-KR mutants. ( f ) Immunoblot analysis to examine the protein expression of WT or ESRP2-KR mutants overexpressed in HEK293T cells. ( g ) qRT–PCR analysis of ENAH exon 11a expression in HEK293T cells transiently transfected with WT or ESRP2-KR mutants upon Arkadia knockdown. Data were normalized to total ENAH. Error bars indicate s.d. siNC, negative control siRNA. Experiments were repeated, and a representative set of data are shown in ( g ). ( h ) Schematic representation of the regulation of ESRP2 function by Arkadia through ubiquitination.
    Figure Legend Snippet: Modulation of the splicing function of ESRP2 by Arkadia through ubiquitination. ( a and b ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( a ) or ESRP2 ( b ), Arkadia wild-type or CA mutant, and ubiquitin. IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin; WT, wild-type. ( c and d ) Immunoprecipitation assay using HEK293T cells transiently transfected with ESRP1 ( c ) or ESRP2 ( d ), WT Arkadia, and ubiquitin WT or its mutants. 7KR: all seven lysine residues are substituted by arginine residues; 6, 11, 27, 29, 33, 48 and 63K: one lysine residue is intact, but the others are substituted by arginine residues. KR, lysine-to-arginine mutation. ( e ) Schematic representation of ESRP2-KR mutants. ( f ) Immunoblot analysis to examine the protein expression of WT or ESRP2-KR mutants overexpressed in HEK293T cells. ( g ) qRT–PCR analysis of ENAH exon 11a expression in HEK293T cells transiently transfected with WT or ESRP2-KR mutants upon Arkadia knockdown. Data were normalized to total ENAH. Error bars indicate s.d. siNC, negative control siRNA. Experiments were repeated, and a representative set of data are shown in ( g ). ( h ) Schematic representation of the regulation of ESRP2 function by Arkadia through ubiquitination.

    Techniques Used: Immunoprecipitation, Transfection, Mutagenesis, Expressing, Quantitative RT-PCR, Negative Control

    21) Product Images from "Stress signaling by Tec tyrosine kinase in the ischemic myocardium"

    Article Title: Stress signaling by Tec tyrosine kinase in the ischemic myocardium

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00273.2010

    Proteomic dissection of Tec tyrosine kinase signaling. A : Tec-associated proteins were purified by FLAG tag-based chromatography ( left ) or immunoprecipitation (IP; right ). Cells transfected with nontagged Tec (NT) were used as a negative control. Proteins were separated by SDS-PAGE, and gels were stained with SYPRO. B : workflow for proteomic analyses. Human embryonic kidney-293 cells were transfected with pcDNA3 containing the Tec-COOH-FLAG insert (or pcDNA3 with untagged Tec as a control). Forty-eight hours later, cells were lysed and subjected to either FLAG IP using anti-FLAG-M2 antibody or column purification with FLAG affinity beads. Tec-associated proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein bands were excised, digested with trypsin, and analyzed with liquid chromotography (LC)/mass spectrometry (MS)/MS on an Orbi-trap. Protein identification was carried out by database searching using the Sequest algorithm. Results from both purification processes were merged and then filtered.
    Figure Legend Snippet: Proteomic dissection of Tec tyrosine kinase signaling. A : Tec-associated proteins were purified by FLAG tag-based chromatography ( left ) or immunoprecipitation (IP; right ). Cells transfected with nontagged Tec (NT) were used as a negative control. Proteins were separated by SDS-PAGE, and gels were stained with SYPRO. B : workflow for proteomic analyses. Human embryonic kidney-293 cells were transfected with pcDNA3 containing the Tec-COOH-FLAG insert (or pcDNA3 with untagged Tec as a control). Forty-eight hours later, cells were lysed and subjected to either FLAG IP using anti-FLAG-M2 antibody or column purification with FLAG affinity beads. Tec-associated proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein bands were excised, digested with trypsin, and analyzed with liquid chromotography (LC)/mass spectrometry (MS)/MS on an Orbi-trap. Protein identification was carried out by database searching using the Sequest algorithm. Results from both purification processes were merged and then filtered.

    Techniques Used: Dissection, Purification, FLAG-tag, Chromatography, Immunoprecipitation, Transfection, Negative Control, SDS Page, Staining, Mass Spectrometry

    22) Product Images from "Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase"

    Article Title: Beclin1 restricts RNA virus infection in plants through suppression and degradation of the viral polymerase

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03658-2

    NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody
    Figure Legend Snippet: NbBeclin1 interacts with NIb. a Yeast-two hybrid (Y2H) assays for possible interactions between NbBeclin1 and each of the 11 TuMV proteins. NbBeclin1 and 11 viral proteins were fused with a GAL4 activation domain (AD-NbBeclin1) and a GAL4-binding domain (BD-P1, BD-HC-Pro, BD-P3, BD-P3N-PIPO, BD-6K1, BD-CI, BD-6K2, BD-NIa-VPg, BD-NIa-Pro, BD-NIb, BD-CP), respectively. Y2H Gold yeast cells co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Trp, -Leu, -His, -Ade or SD/-Trp, -Leu medium to screen for positive interactions at 3 days after transformation. Yeast co-transformed with AD-T7-T+BD-T7-53 serves as a positive control; yeast cells co-transformed with AD-NbBeclin1 and the empty BD or with the empty AD and BD-NIb are negative controls. b BiFC assays between NbBeclin1 and NIb in the leaves of H2B-RFP transgenic N . benthamiana . Confocal imaging was performed at 48 hpi. NbBeclin1 and NIb were fused to the N (YN) and C-terminal (YC) fragments of yellow fluorescent protein (YFP). The NbBeclin1-NIb interaction led to the reconstituted fluorescence-competent structure and restoration of yellow fluorescence (green). Nuclei of tobacco leaf epidermal cells are indicated by the expression of H2B-RFP transgene (red). Bars, 50 μm. c Co-localization of NIb-YFP with NbBeclin1-CFP in the leaf cells of H2B-RFP transgenic N . benthamiana by confocal microscopy at 48 hpi. Arrow indicates yellow fluorescence, which was produced from the overlapping of NIb-YFP (green) and NbBeclin1-CFP (red). Bars, 50 μm. d Co-immunoprecipitation (Co-IP) analysis of NbBeclin1-CFP and Myc-NIb in vivo. N . benthamiana leaves were co-infiltrated with A . tumefaciens cells harboring expression vectors to express NbBeclin1-CFP and Myc-NIb (Lane 1), NbBeclin1-CFP and Myc-P3N-PIPO (Lane 2), Myc-NIb and GFP (Lane 3), and GFP and Myc-P3N-PIPO (Lane 4). Leaf protein extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody

    Techniques Used: Activation Assay, Binding Assay, Transformation Assay, Positive Control, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Imaging, Fluorescence, Expressing, Confocal Microscopy, Produced, Immunoprecipitation, Co-Immunoprecipitation Assay, In Vivo, Incubation, Immu-Puri

    23) Product Images from "ccm2-like is required for cardiovascular development as a novel component of the Heg-CCM pathway"

    Article Title: ccm2-like is required for cardiovascular development as a novel component of the Heg-CCM pathway

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2013.01.006

    Biochemical interactions among proteins of the Heg-CCM pathway. (A) Schematic of all Ccm1 constructs used for co-immunoprecipitation experiments. (B) FLAG-ccm1 and FLAG-ccm1ΔFERM co-immunoprecipitate HA-ccm2 and HA-ccm2l, while the N-terminal Ccm1 deletion proteins do not bind HA-ccm2 and HA-ccm2l. In contrast, FLAG-ccm1ΔFERM does not co-immunoprecipitate Heg, while the N-terminal Ccm1 deletion proteins do. (C) HA-ccm2 binding to FLAG-ccm1 is severely weakened by mutation of Ccm1’s two NPXY motifs and further disrupted by mutations in the NPXF motif. The strength of the interaction between HA-ccm2l and FLAG-ccm1 is unaffected by mutation of Ccm1’s NPXY motifs but is severely diminished when all three NPXY/F motifs are mutated. Heg-Ccm1 interactions appear unaffected by mutation of either two or all three NPXY/F motifs in Ccm1. (D) The mutant Ccm1 proteins FLAG-ccm1ty219c and FLAG-ccm1m775 bind HA-ccm2 and HA-ccm2l as well as wildtype FLAG-ccm1 does, but they do not bind Heg.
    Figure Legend Snippet: Biochemical interactions among proteins of the Heg-CCM pathway. (A) Schematic of all Ccm1 constructs used for co-immunoprecipitation experiments. (B) FLAG-ccm1 and FLAG-ccm1ΔFERM co-immunoprecipitate HA-ccm2 and HA-ccm2l, while the N-terminal Ccm1 deletion proteins do not bind HA-ccm2 and HA-ccm2l. In contrast, FLAG-ccm1ΔFERM does not co-immunoprecipitate Heg, while the N-terminal Ccm1 deletion proteins do. (C) HA-ccm2 binding to FLAG-ccm1 is severely weakened by mutation of Ccm1’s two NPXY motifs and further disrupted by mutations in the NPXF motif. The strength of the interaction between HA-ccm2l and FLAG-ccm1 is unaffected by mutation of Ccm1’s NPXY motifs but is severely diminished when all three NPXY/F motifs are mutated. Heg-Ccm1 interactions appear unaffected by mutation of either two or all three NPXY/F motifs in Ccm1. (D) The mutant Ccm1 proteins FLAG-ccm1ty219c and FLAG-ccm1m775 bind HA-ccm2 and HA-ccm2l as well as wildtype FLAG-ccm1 does, but they do not bind Heg.

    Techniques Used: Construct, Immunoprecipitation, Binding Assay, Mutagenesis

    24) Product Images from "The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division"

    Article Title: The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.14.6379-6392.2004

    The S. pombe Nup107-120 NPC subcomplex biochemically purifies as two distinct entities. (A) Western blot analysis of immunoprecipitation experiments performed on S. pombe whole-cell lysates using an anti-GFP antibody. S. pombe strains expressing an HA-Nup133a, -Nup133b, or -Nup120 fusion in a Δ nup133a , Δ nup133b , or Δ nup120 background, respectively, and either no GFP fusion or a GFP-tagged nucleoporin (Nup107-GFP, GFP-Nup85, or GFP-Seh1) were grown at 30°C to log phase in supplemented liquid EMM in the presence of thiamine. Immunoprecipitation experiments were performed on these 12 different S. pombe whole-cell lysates using an anti-GFP antibody. Equivalent amounts of total protein extracts (T), depleted supernatants (S), and a 10-fold equivalent of the immune pellets (P) were analyzed by Western blotting using anti-GFP (a) or anti-HA (a′, b′, and c′) antibody. The results with the GFP antibody were similar in all experiments and are presented only for the strain expressing HA-Nup133a. Molecular mass markers are on the right in kilodaltons. (B) Silver staining of immunoprecipitates from control, GFP-SpNup85, GFP-Seh1, and Nup107-GFP cell extracts obtained with an anti-GFP antibody. The asterisks indicate the position of the GFP fusion in each lane. The dots indicate additional bands subsequently identified by mass spectrometry as Nup145-C (band 1) and Nup85 (band 2). (C) Schematic model of the Nup107-120 complex in S. pombe , based on the structural data obtained from S. cerevisiae ).
    Figure Legend Snippet: The S. pombe Nup107-120 NPC subcomplex biochemically purifies as two distinct entities. (A) Western blot analysis of immunoprecipitation experiments performed on S. pombe whole-cell lysates using an anti-GFP antibody. S. pombe strains expressing an HA-Nup133a, -Nup133b, or -Nup120 fusion in a Δ nup133a , Δ nup133b , or Δ nup120 background, respectively, and either no GFP fusion or a GFP-tagged nucleoporin (Nup107-GFP, GFP-Nup85, or GFP-Seh1) were grown at 30°C to log phase in supplemented liquid EMM in the presence of thiamine. Immunoprecipitation experiments were performed on these 12 different S. pombe whole-cell lysates using an anti-GFP antibody. Equivalent amounts of total protein extracts (T), depleted supernatants (S), and a 10-fold equivalent of the immune pellets (P) were analyzed by Western blotting using anti-GFP (a) or anti-HA (a′, b′, and c′) antibody. The results with the GFP antibody were similar in all experiments and are presented only for the strain expressing HA-Nup133a. Molecular mass markers are on the right in kilodaltons. (B) Silver staining of immunoprecipitates from control, GFP-SpNup85, GFP-Seh1, and Nup107-GFP cell extracts obtained with an anti-GFP antibody. The asterisks indicate the position of the GFP fusion in each lane. The dots indicate additional bands subsequently identified by mass spectrometry as Nup145-C (band 1) and Nup85 (band 2). (C) Schematic model of the Nup107-120 complex in S. pombe , based on the structural data obtained from S. cerevisiae ).

    Techniques Used: Western Blot, Immunoprecipitation, Expressing, Silver Staining, Mass Spectrometry

    25) Product Images from "Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma"

    Article Title: Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma

    Journal: Oncogenesis

    doi: 10.1038/s41389-018-0050-x

    TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Binding Assay, Immunoprecipitation, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

    26) Product Images from "Membrane protein recycling from the vacuole/lysosome membrane"

    Article Title: Membrane protein recycling from the vacuole/lysosome membrane

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201709162

    Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.
    Figure Legend Snippet: Specific residues in the cytoplasmic tail of Atg27 are required for its retrograde traffic. (A) Schematic of Atg27 mutational analysis from Fig. S3 C. (B) Atg27-diL-GFP localization after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-diL-GFP on the vacuole in B. (D) Atg27-diL-GFP mutant localization in vps35 Δ cells after 1 h of rapamycin treatment. (E) The percentage of cells with Atg27-diL-GFP on the vacuole in D and Fig. S3 D. (F) Cells were treated with rapamycin for 1 h and Atg27-diL immunoprecipitated. Interacting Snx4-3XHA mutants were detected by immunoblotting. (G) Model for Atg27 trafficking. Atg27 is delivered to the vacuole membrane via the AP-3 pathway and then recycled from the vacuole membrane to the endosome by the Snx4 complex. Atg27 is then retrieved from the endosome by retromer. (H) Membrane trafficking pathways in yeast cells. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation. Bars, 2 µm.

    Techniques Used: Mutagenesis, Immunoprecipitation, Two Tailed Test

    The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.
    Figure Legend Snippet: The Snx4 complex mediates Atg27 vacuole-to-endosome retrograde transport. (A) Schematic predicting Atg27 accumulation on the vacuole membrane in retromer and recycling double mutants. (B) Atg27-GFP localization in vps35 Δ snx4 Δ cells after 1 h of rapamycin treatment. (C) The percentage of cells with Atg27-GFP on the vacuole. (D) Atg27-GFP localization in vps35ts snx4 Δ mutants after shift to 37°C with rapamycin treatment for 1 h. (E) Quantification of Atg27-GFP fluorescence intensity on the vacuole from D and Fig. S2 C. (F) Cells were treated with rapamycin for 1 h, Atg27-GFP was immunoprecipitated, and interacting Snx4-3XHA was detected by immunoblot. For all quantification shown in this figure, at least 100 cells were measured, and the data from three independent experiments were used for statistical analysis; two-tailed Student’s t test. Error bars represent SD. IP, immunoprecipitation; n.s., not significant. Bar, 2 µm.

    Techniques Used: Fluorescence, Immunoprecipitation, Two Tailed Test

    27) Product Images from "African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell"

    Article Title: African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.24.12473-12482.2002

    Gel mapping, stoichiometry, and relative abundances of ASFV polyprotein products in the virus particle. (A) 2-D gel electrophoresis of ASFV labeled with [ 35 S]methionine. Basic proteins were resolved by NEPHGE, while acidic proteins were separated by IEF. The positions of the major capsid protein p72; the polyprotein pp220-derived products p150, p37, p34, and p14; and the polyprotein pp62-derived products p35 and p15 are indicated. The migrations of molecular weight (MW) markers (in thousands) and of pI markers are indicated at the right and at the bottom of the 2-D gels, respectively. (B) 2-D mapping of proteins p35 and p15. Protein p15 was mapped in NEPHGE gels by immunoprecipitation, whereas p35 was identified in IEF gels by immunoblotting. (C) 1-D gel electrophoresis of ASFV particles labeled with [ 35 S]methionine or 14 C-amino acids or stained with Coomassie blue (C.B.). The positions of several structural proteins are indicated. (D) Quantitative analysis of the polyprotein products. Polyprotein products and, as a reference, capsid protein p72 were quantified by densitometry analysis of 2-D gels of [ 35 S]methionine-labeled proteins (two experiments) or of 1-D gels of ASFV proteins labeled with [ 35 S]methionine (three experiments) or 14 C-amino acids (two experiments) or stained with Coomassie blue (three experiments). Stoichiometry data were normalized by the number of methionine residues of each protein (for 35 S-labeled proteins) or by its molecular weight (for 14 C-labeled or Coomassie blue-stained proteins) and referred to protein p34. Relative abundances (percentages) were estimated from 1-D gels of 14 C-labeled or Coomassie blue-stained proteins. Standard deviations of the means were less than 25% of the means. Proteins p150 and p72 were not quantified in 2-D gels due to deficient focusing, while proteins p15 and p14 were not estimated in 1-D gels because of heterogeneity of the bands. The relative abundances of proteins p15 and p14 are values extrapolated by assuming an equimolar stoichiometry. Stoichiometry data for p37 and p14 [marked with asterisks in the 35 .
    Figure Legend Snippet: Gel mapping, stoichiometry, and relative abundances of ASFV polyprotein products in the virus particle. (A) 2-D gel electrophoresis of ASFV labeled with [ 35 S]methionine. Basic proteins were resolved by NEPHGE, while acidic proteins were separated by IEF. The positions of the major capsid protein p72; the polyprotein pp220-derived products p150, p37, p34, and p14; and the polyprotein pp62-derived products p35 and p15 are indicated. The migrations of molecular weight (MW) markers (in thousands) and of pI markers are indicated at the right and at the bottom of the 2-D gels, respectively. (B) 2-D mapping of proteins p35 and p15. Protein p15 was mapped in NEPHGE gels by immunoprecipitation, whereas p35 was identified in IEF gels by immunoblotting. (C) 1-D gel electrophoresis of ASFV particles labeled with [ 35 S]methionine or 14 C-amino acids or stained with Coomassie blue (C.B.). The positions of several structural proteins are indicated. (D) Quantitative analysis of the polyprotein products. Polyprotein products and, as a reference, capsid protein p72 were quantified by densitometry analysis of 2-D gels of [ 35 S]methionine-labeled proteins (two experiments) or of 1-D gels of ASFV proteins labeled with [ 35 S]methionine (three experiments) or 14 C-amino acids (two experiments) or stained with Coomassie blue (three experiments). Stoichiometry data were normalized by the number of methionine residues of each protein (for 35 S-labeled proteins) or by its molecular weight (for 14 C-labeled or Coomassie blue-stained proteins) and referred to protein p34. Relative abundances (percentages) were estimated from 1-D gels of 14 C-labeled or Coomassie blue-stained proteins. Standard deviations of the means were less than 25% of the means. Proteins p150 and p72 were not quantified in 2-D gels due to deficient focusing, while proteins p15 and p14 were not estimated in 1-D gels because of heterogeneity of the bands. The relative abundances of proteins p15 and p14 are values extrapolated by assuming an equimolar stoichiometry. Stoichiometry data for p37 and p14 [marked with asterisks in the 35 .

    Techniques Used: Nucleic Acid Electrophoresis, Labeling, Electrofocusing, Derivative Assay, Molecular Weight, Immunoprecipitation, Staining

    28) Product Images from "A function-blocking CD47 antibody suppresses stem cell and EGF signaling in triple-negative breast cancer"

    Article Title: A function-blocking CD47 antibody suppresses stem cell and EGF signaling in triple-negative breast cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7100

    A. AEGFR-immunoprecipitation from MDA-MB-231 cell extracts followed by western blotting shows that B6H12 treatment for 15 min disrupts the association between EGFR and CD47 and inhibits EGFR-Y 1068 phosphorylation. B . CD47-immunoprecipitation showed that a fraction of EGFR co-immunoprecipitates with EGFR. B6H12 treatment for 15 min reduced interaction between CD47 and EGFR in MDA-MB-231 cells. ( C .- D .) MDA-MB-231 cells were pretreated with B6H12 for 15 minutes followed by EGF for 5 minutes, and IP-western blotting was performed using phospho-EGFR antibody. D . Quantification of three experiments was analyzed using the t-test (*P
    Figure Legend Snippet: A. AEGFR-immunoprecipitation from MDA-MB-231 cell extracts followed by western blotting shows that B6H12 treatment for 15 min disrupts the association between EGFR and CD47 and inhibits EGFR-Y 1068 phosphorylation. B . CD47-immunoprecipitation showed that a fraction of EGFR co-immunoprecipitates with EGFR. B6H12 treatment for 15 min reduced interaction between CD47 and EGFR in MDA-MB-231 cells. ( C .- D .) MDA-MB-231 cells were pretreated with B6H12 for 15 minutes followed by EGF for 5 minutes, and IP-western blotting was performed using phospho-EGFR antibody. D . Quantification of three experiments was analyzed using the t-test (*P

    Techniques Used: Immunoprecipitation, Multiple Displacement Amplification, Western Blot

    29) Product Images from "Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells"

    Article Title: Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003821

    Nuclear Ago1 interacts with RNAP II. ( A ) Immunoprecipitation (IP) assays were performed on nuclear extracts from PC-3 cells using Ago1 or Ago2 antibodies. IgG served as a negative IP control. Immunoprecipitates were analyzed by immunoblotting (IB) with the indicated antibodies. RNAP II and Tubulin were also detected by IB analysis to validate nuclear (Nuc) and cytoplasmic (Cyto) fractions. ( B ) Reciprocal IP analysis was performed on nuclear extracts from PC-3 cells using an antibody specific to RNAP II. IB detected pulldown of Ago1 and RNAP II but not Ago2. Input control represents 10% nuclear extract used for IP. * denotes a nonspecific band. ( C ) IP was performed on nuclear extracts from LNCaP cells as in (A). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were confirmed by IB analysis.
    Figure Legend Snippet: Nuclear Ago1 interacts with RNAP II. ( A ) Immunoprecipitation (IP) assays were performed on nuclear extracts from PC-3 cells using Ago1 or Ago2 antibodies. IgG served as a negative IP control. Immunoprecipitates were analyzed by immunoblotting (IB) with the indicated antibodies. RNAP II and Tubulin were also detected by IB analysis to validate nuclear (Nuc) and cytoplasmic (Cyto) fractions. ( B ) Reciprocal IP analysis was performed on nuclear extracts from PC-3 cells using an antibody specific to RNAP II. IB detected pulldown of Ago1 and RNAP II but not Ago2. Input control represents 10% nuclear extract used for IP. * denotes a nonspecific band. ( C ) IP was performed on nuclear extracts from LNCaP cells as in (A). Nuclear (Nuc) and cytoplasmic (Cyto) fractions were confirmed by IB analysis.

    Techniques Used: Immunoprecipitation

    30) Product Images from "Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma"

    Article Title: Dysregulation of the MiR-449b target TGFBI alters the TGFβ pathway to induce cisplatin resistance in nasopharyngeal carcinoma

    Journal: Oncogenesis

    doi: 10.1038/s41389-018-0050-x

    TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P
    Figure Legend Snippet: TGFBI regulates signaling pathways by binding ITGB3 and ITGB5. a ITGB3 immunoprecipitation (IP: αMyc) was performed on HEK293T cells expressing ITGB3 in the absence or the presence of TGFBI (via transfection). Interaction between ITGB3 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. b ITGB5 immunoprecipitation (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC with or without presence of TGFBI (via transfection). Interaction between ITGB5 and TGFBI was detected using anti-TGFBI (αTGFBI) antibody. c Immunoprecipitation of pro-TGFβ1 (IP: αFlag) was performed on HEK293T cells expressing pro-TGFβ1 alone or with ITGB3. Interaction between ITGB3 and pro-TGFβ1 was identified using anti-Myc (αMyc) antibody. d Immunoprecipitation of ITGB5 (IP: αGFP) was performed on HEK293T cells expressing ITGB5ΔC alone or with pro-TGFβ1. Interaction between ITGB5 and pro-TGFβ1 was revealed using anti-Flag (αFlag) antibody. e Co-immunoprecipitations were performed on HEK293T cells expressing ITGB3 alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB3 binding. f Co-immunoprecipitations were performed on HEK293T expressing ITGB5ΔC alone or with pro-TGFβ1 in the absence (TGFβ1-Flag) or presence of TGFBI (TGFβ1-Flag + TGFBI). These data show competition between TGFBI and pro-TGFβ1 for ITGB5 binding. For a–f Western blot of the whole-cell lysates (WCLs) before pull-down is shown as a control for specificity and expression. g Top: relative luciferase activity was assessed after transient transfection of pro-TGFβ1-Flag and TGFBI-Myc, as indicated, and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot of HEK293T cells transfected with pro-TGFβ1-Flag encoding plasmid (1 µg) and TGFBI-Myc encoding plasmid, as indicated. Anti-Myc (αMyc) antibody was used to assess the expression of TGFBI, and anti-Flag (αFlag) antibody was used to assess the expression of pro-TGFβ1 and active TGFβ1 forms. The expression of pro-TGFβ1 indicates similar transfection efficiency across the conditions. Anti-β-actin (αβ-actin) antibody was used as the loading control. h Top: relative luciferase activity was assessed after transient transfection of ITGB3-Myc (1 µg) or ITGB5ΔC-GFP (1 µg) and TGFBI (2 µg), and the co-transfection of pSBE4-luciferase vector (150 ng) and Renilla plasmid (100 ng). Bottom: western blot control of the plasmids expression: anti-TGFBI (αTGFBI) antibody was used to assess the expression of TGFBI, anti-Myc (αMyc), and anti-GFP (αGFP) for ITGB3 and ITGB5, respectively. Anti-β-actin (αβ-actin) antibody was used as the loading control. The data are expressed as the mean ± SEM of at least three independent experiments. * P

    Techniques Used: Binding Assay, Immunoprecipitation, Expressing, Transfection, Western Blot, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation

    31) Product Images from "PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner"

    Article Title: PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

    Journal: Cell Research

    doi: 10.1038/cr.2014.34

    Notch1 interacts with PKCζ at the membrane. (A) Untransfected C2C12 cells undergoing differentiation were harvested at different time points and subjected to immunoprecipitation (IP) using a Notch1 antibody (C20 goat). Immunoblotting was performed
    Figure Legend Snippet: Notch1 interacts with PKCζ at the membrane. (A) Untransfected C2C12 cells undergoing differentiation were harvested at different time points and subjected to immunoprecipitation (IP) using a Notch1 antibody (C20 goat). Immunoblotting was performed

    Techniques Used: Immunoprecipitation

    32) Product Images from "The testis-specific LINC component SUN3 is essential for sperm head shaping during mouse spermiogenesis"

    Article Title: The testis-specific LINC component SUN3 is essential for sperm head shaping during mouse spermiogenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.012375

    SUN3 interacts with SUN4 in mouse testes, and SUN4 protein levels are drastically reduced in Sun3 −/− mice. A , Western blotting of immunoprecipitated testis extract showing the presence of SUN3 proteins in lysates immunoprecipitated by rabbit anti-SUN3 antibodies. Rabbit IgG was used as a negative control. B , Western blotting showing the presence of SUN4 proteins in testis lysates immunoprecipitated by guinea pig anti-SUN3 antibodies. Rabbit IgG was used as a negative control. IP , immunoprecipitation; WB , Western blotting. C , immunofluorescence staining of SUN4 ( red ), SP56 ( green ), and Hoechst ( blue ) on testis cell spreads from 8-week-old Sun3 +/+ and Sun3 −/− mice, showing mislocalization of SUN4 in Sun3 −/− spermatids. Scale bars = 20 μm. D , Western blotting showing the levels of SUN4 proteins in Sun3 +/+ and Sun3 −/− testes. β-Actin was used as a loading control. For A , B , and D , the bands corresponding to the sizes of proteins of interest are indicated by arrows .
    Figure Legend Snippet: SUN3 interacts with SUN4 in mouse testes, and SUN4 protein levels are drastically reduced in Sun3 −/− mice. A , Western blotting of immunoprecipitated testis extract showing the presence of SUN3 proteins in lysates immunoprecipitated by rabbit anti-SUN3 antibodies. Rabbit IgG was used as a negative control. B , Western blotting showing the presence of SUN4 proteins in testis lysates immunoprecipitated by guinea pig anti-SUN3 antibodies. Rabbit IgG was used as a negative control. IP , immunoprecipitation; WB , Western blotting. C , immunofluorescence staining of SUN4 ( red ), SP56 ( green ), and Hoechst ( blue ) on testis cell spreads from 8-week-old Sun3 +/+ and Sun3 −/− mice, showing mislocalization of SUN4 in Sun3 −/− spermatids. Scale bars = 20 μm. D , Western blotting showing the levels of SUN4 proteins in Sun3 +/+ and Sun3 −/− testes. β-Actin was used as a loading control. For A , B , and D , the bands corresponding to the sizes of proteins of interest are indicated by arrows .

    Techniques Used: Mouse Assay, Western Blot, Immunoprecipitation, Negative Control, Immunofluorescence, Staining

    33) Product Images from "Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles"

    Article Title: Nuclear Export and Import of Human Hepatitis B Virus Capsid Protein and Particles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001162

    Specific physical and functional interactions between a cellular TAP protein and HBc ARD were shown by experiments of co-immunoprecipitation, si-RNA treatment, and cotransfection with a CMV-TAP expression vector. Fig. 7A . Huh7 cells were transfected with expression vectors of a) SV40 LT, or b) SV40 LT-HBc ARD chimera. Transfected cell lysates were immunoprecipitated (IP) with anti-SV40 LT antibody. Equal amounts of immunoprecipitated cell lysates from a) or b) were loaded on SDS-PAGE, respectively, followed by Western blot analysis using anti-TAP or anti-SV40 LT antibodies. The 66 kDa TAP protein can be detected only in cell lysates transfected with SV40 LT-HBc ARD, but not transfected with wild type SV40 LT, indicating that TA P could bind to HBc ARD. This result was confirmed in a reciprocal experiment using an anti-TAP antibody for IP. Fig. 7B . Knockdown of endogenous TAP protein by siRNA against TAP ( panel b ) resulted in a more than 17-fold increase of nuclear accumulation of wild type HBc (N > C pattern shifted from 3% to 52%). In contrast, control siRNA (Nontarget) ( panel a ) did not affect the subcellular localization pattern of HBc. HBc (red), α-tubulin (green) and DAPI (blue). Fig. 7C . Treatment of Huh7 cells with TAP-specific siRNA resulted in appreciable reduction of TAP protein by Western blot analysis using anti-TAP antibody. * Non-specific bands served as an internal control. (cf Materials and Methods for detail). Fig. 7D . The nuclear predominant phenotype of wild type HBc in Huh7 cells treated with siTAP can be reverted efficiently to a cytoplasmic predominant phenotype by cotransfection with a CMV-TAP expression vector. Fig. 7E . Western blot analysis of TAP protein in Huh7 cells transfected with a vector only or a plasmid CMV-TAP. Fig. 7F . Upon treatment with siTAP, wild type HBV DNA synthesis in Huh7 cells was reduced approximately 7-fold by Southern blot analysis. RC: relaxed circle DNA, SS: single-strand DNA. While there was no difference in the results of MTT cytotoxicity assay using media from cell culture with or without siTAP treatment, the HBsAg level by ELISA was reduced by approximately 10-fold with siTAP treatment. The averaged values of DNA intensity, MTT assay, and HBsAg assay were processed from four independent experiments ( Materials and Methods ).
    Figure Legend Snippet: Specific physical and functional interactions between a cellular TAP protein and HBc ARD were shown by experiments of co-immunoprecipitation, si-RNA treatment, and cotransfection with a CMV-TAP expression vector. Fig. 7A . Huh7 cells were transfected with expression vectors of a) SV40 LT, or b) SV40 LT-HBc ARD chimera. Transfected cell lysates were immunoprecipitated (IP) with anti-SV40 LT antibody. Equal amounts of immunoprecipitated cell lysates from a) or b) were loaded on SDS-PAGE, respectively, followed by Western blot analysis using anti-TAP or anti-SV40 LT antibodies. The 66 kDa TAP protein can be detected only in cell lysates transfected with SV40 LT-HBc ARD, but not transfected with wild type SV40 LT, indicating that TA P could bind to HBc ARD. This result was confirmed in a reciprocal experiment using an anti-TAP antibody for IP. Fig. 7B . Knockdown of endogenous TAP protein by siRNA against TAP ( panel b ) resulted in a more than 17-fold increase of nuclear accumulation of wild type HBc (N > C pattern shifted from 3% to 52%). In contrast, control siRNA (Nontarget) ( panel a ) did not affect the subcellular localization pattern of HBc. HBc (red), α-tubulin (green) and DAPI (blue). Fig. 7C . Treatment of Huh7 cells with TAP-specific siRNA resulted in appreciable reduction of TAP protein by Western blot analysis using anti-TAP antibody. * Non-specific bands served as an internal control. (cf Materials and Methods for detail). Fig. 7D . The nuclear predominant phenotype of wild type HBc in Huh7 cells treated with siTAP can be reverted efficiently to a cytoplasmic predominant phenotype by cotransfection with a CMV-TAP expression vector. Fig. 7E . Western blot analysis of TAP protein in Huh7 cells transfected with a vector only or a plasmid CMV-TAP. Fig. 7F . Upon treatment with siTAP, wild type HBV DNA synthesis in Huh7 cells was reduced approximately 7-fold by Southern blot analysis. RC: relaxed circle DNA, SS: single-strand DNA. While there was no difference in the results of MTT cytotoxicity assay using media from cell culture with or without siTAP treatment, the HBsAg level by ELISA was reduced by approximately 10-fold with siTAP treatment. The averaged values of DNA intensity, MTT assay, and HBsAg assay were processed from four independent experiments ( Materials and Methods ).

    Techniques Used: Functional Assay, Immunoprecipitation, Cotransfection, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, DNA Synthesis, Southern Blot, MTT Assay, Cytotoxicity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, HBsAg Assay

    34) Product Images from "HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development"

    Article Title: HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

    Journal: eLife

    doi: 10.7554/eLife.28672

    HMMR enables the PLK1-dependent spindle pole positioning pathway. ( A ) Western blot analysis of mitotic HeLa extracts stably expressing DHC-GFP treated with scrambled (siScr) siRNA or siRNA targeting HMMR (siHMMR) subjected to immunoprecipitation with GFP antibody or control IgG and blotted with the indicated antibodies, including GFP (GFP-DHC), dynein intermediate chain (DIC), p150 glued , DYNLL1, HMMR or Actin (four replicates). ( B ) Localization of Flag-Dynein light chain (DYNLL1) in HeLa cells treated with siScr or siHMMR (three replicates). Scale bars, 10 μm. ( C ) Localization of CHICA in cells treated with siScr and siHMMR (three replicates). Scale bars, 10 μm. ( D ) Localization of phospho-PLK1 (pPLK1) in HeLa cells expressing siScr or siHMMR. Scale bars, 5 μm. ( E ) Quantification of pPLK1 intensity from pole to pole. Data represents mean (50 cells from three experiments). ( F ) Astral microtubules contact the cortex in cells treated with siScr, siHMMR, or BI2536 (Plk1 inhibitor). Yellow box indicates EB1 inset and white dotted line indicates region of quantification. Scale bar, 5 μm. ( G ) Quantification of EB1 at the cortex in cells treated with siScr, siHMMR, or BI2536. Data are represented as mean ±SD (***p
    Figure Legend Snippet: HMMR enables the PLK1-dependent spindle pole positioning pathway. ( A ) Western blot analysis of mitotic HeLa extracts stably expressing DHC-GFP treated with scrambled (siScr) siRNA or siRNA targeting HMMR (siHMMR) subjected to immunoprecipitation with GFP antibody or control IgG and blotted with the indicated antibodies, including GFP (GFP-DHC), dynein intermediate chain (DIC), p150 glued , DYNLL1, HMMR or Actin (four replicates). ( B ) Localization of Flag-Dynein light chain (DYNLL1) in HeLa cells treated with siScr or siHMMR (three replicates). Scale bars, 10 μm. ( C ) Localization of CHICA in cells treated with siScr and siHMMR (three replicates). Scale bars, 10 μm. ( D ) Localization of phospho-PLK1 (pPLK1) in HeLa cells expressing siScr or siHMMR. Scale bars, 5 μm. ( E ) Quantification of pPLK1 intensity from pole to pole. Data represents mean (50 cells from three experiments). ( F ) Astral microtubules contact the cortex in cells treated with siScr, siHMMR, or BI2536 (Plk1 inhibitor). Yellow box indicates EB1 inset and white dotted line indicates region of quantification. Scale bar, 5 μm. ( G ) Quantification of EB1 at the cortex in cells treated with siScr, siHMMR, or BI2536. Data are represented as mean ±SD (***p

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

    35) Product Images from "Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus"

    Article Title: Drosophila TRP channels require a protein with a distinctive motif encoded by the inaF locus

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

    doi: 10.1073/pnas.0708368104

    Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.
    Figure Legend Snippet: Association of HA-tagged INAF-PB with TRP channels. ( A ) Immunolocalization. A representative confocal section of retina, costained with anti-TRP and anti-HA, from adult flies bearing the tagged version of inaF-PB , is shown. To eliminate interference by eye pigment, the transgenic line also carried bw and st mutations. Within the ommatidium shown, signal from INAF-B is detected in rhabdomeres (and not their surrounding cell bodies) and colocalizes with signal from TRP. ( B–F ) Immunoprecipitation. The top line of the key for each panel shows the antibody used to precipitate dodecyl-β-maltoside extracts of fly heads: anti-HA (H), anti-TRP (T), anti-INAD (D), or a nonspecific control antibody (N). The second line of the key indicates the presence (+) or absence (−) of an HA tag on a transgenic copy of INAF-B. Where appropriate, the key also indicates the wild-type (+) or mutant (−) status of the locus whose genotype varies in the samples of the panel. Immunoprecipitates were electrophoresed and then probed with the antibody indicated at the left of each panel. The lanes with IP status marked (−) contain a sample (≈3%) of material used as input for the corresponding immunoprecipitations.

    Techniques Used: Transgenic Assay, Immunoprecipitation, Mutagenesis

    36) Product Images from "Mechanisms of Human Papillomavirus Type 16 Neutralization by L2 Cross-Neutralizing and L1 Type-Specific Antibodies "

    Article Title: Mechanisms of Human Papillomavirus Type 16 Neutralization by L2 Cross-Neutralizing and L1 Type-Specific Antibodies

    Journal: Journal of Virology

    doi: 10.1128/JVI.00143-08

    Immunoprecipitation of L1/L2 complexes. The anti-L1 MAb H16.V5 (lanes 1 and 3) or anti-L2 MAb RG-1 (lanes 2 and 4) was used to immunoprecipitate either L1- and L2-containing mature pseudovirions (lanes 1 and 2) or crude lysates that contained a mixture of L1 and L2 assembly intermediates and immature pseudovirions (lanes 3 and 4). The immunoblot was reacted with a biotinylated anti-L1 monoclonal antibody. The arrow indicates the migration of L1. The IgG heavy chain is the lower heavy band.
    Figure Legend Snippet: Immunoprecipitation of L1/L2 complexes. The anti-L1 MAb H16.V5 (lanes 1 and 3) or anti-L2 MAb RG-1 (lanes 2 and 4) was used to immunoprecipitate either L1- and L2-containing mature pseudovirions (lanes 1 and 2) or crude lysates that contained a mixture of L1 and L2 assembly intermediates and immature pseudovirions (lanes 3 and 4). The immunoblot was reacted with a biotinylated anti-L1 monoclonal antibody. The arrow indicates the migration of L1. The IgG heavy chain is the lower heavy band.

    Techniques Used: Immunoprecipitation, Migration

    37) Product Images from "Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP"

    Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.193

    Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand
    Figure Legend Snippet: Cab45S interacts with the nucleotide-binding domain (NBD) of GRP78/BiP. ( a ) Immunoprecipitation assay (IP) with anti-Flag antibody in HEK293T cell lysates expressing 3 × Flag-Cab45S. Immunoprecipitates were subjected to SDS-PAGE and then MS analysis. ( b ) Extracts of HEK293T cells co-transfected with GRP78/BiP-EGFP and 3 × Flag, 3 × Flag-Cab45S, 3 × Flag-Cab45G or 3 × Flag-RCN1 were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibody. ( c and d ) Mapping the domain at which GRP78/BiP interacted with Cab45S. Schematics of GRP78/BiP truncates ( c ). Extracts of HEK293T cells overexpressing GFP-tagged GRP78/BiP truncates and 3 × Flag-Cab45S were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibody ( d ). SBD, substrate-binding domain. ( e and f ) Mapping the domain of Cab45S, which interacted with GRP78/BiP. Schematics of Cab45S truncates ( e ). Extracts of HEK293T cells overexpressing 3 × Flag-tagged Cab45S truncates and GRP78/BiP-EGFP were immunoprecipitated with anti-Flag antibody, and the immunoprecipitates were immunoblotted with anti-GFP and anti-Flag antibodies ( f ). Asterisks indicate 3 × Flag-tagged Cab45S truncates immunoprecipitated by anti-Flag antibody. SP, signal peptide; EFh, EF-hand

    Techniques Used: Binding Assay, Immunoprecipitation, Expressing, SDS Page, Mass Spectrometry, Transfection

    38) Product Images from "The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP"

    Article Title: The lysosomal transporter MFSD1 is essential for liver homeostasis and critically depends on its accessory subunit GLMP

    Journal: eLife

    doi: 10.7554/eLife.50025

    Screening of different detergents for the MFSD1-GLMP-interaction by co-immunoprecipitation and additional controls for FACS-based FRET and immunoprecipitation. ( A ) Co-immunoprecipitation of untagged MFSD1 and HA-tagged GLMP from transfected HeLa cell lysates solubilized with different detergents with antibody against MFSD1. ( B ) Co-immunoprecipitation of C-terminal GFP-tagged MFSD1 and HA-tagged GLMP from transfected HeLa cell lysates solubilized with CHAPS. MFSD1-GFP was precipitated using an antibody against GFP or an unspecific control antibody (cAb). HA-tagged GLMP and LAMP1 (included as a negative control) were detected by immunoblot with an antibody against HA. LIMP2 was detected as an additional control and was exclusively detected in the lysates, but not the GFP-precipitate. ( C ) Co-immunoprecipitation of C-terminal GFP-tagged MFSD1 LL/AA PM mutant and HA-tagged GLMP Y400A from transfected HeLa cell lysates solubilized with CHAPS. MFSD1 LL/AA -GFP was precipitated using an antibody against GFP or an unspecific control antibody (cAb). HA-tagged GLMP Y400A and LAMP1 Y414A (PM localized LAMP1 variant included as a negative control) were detected by immunoblot with an antibody against HA. ( D ) Plot of the GFP intensity of the HeLa cells transfected with different plasmids used for the flow cytometry-based FRET analysis. The number in the plot represents the percentage of GFP+ cells alive for each condition.
    Figure Legend Snippet: Screening of different detergents for the MFSD1-GLMP-interaction by co-immunoprecipitation and additional controls for FACS-based FRET and immunoprecipitation. ( A ) Co-immunoprecipitation of untagged MFSD1 and HA-tagged GLMP from transfected HeLa cell lysates solubilized with different detergents with antibody against MFSD1. ( B ) Co-immunoprecipitation of C-terminal GFP-tagged MFSD1 and HA-tagged GLMP from transfected HeLa cell lysates solubilized with CHAPS. MFSD1-GFP was precipitated using an antibody against GFP or an unspecific control antibody (cAb). HA-tagged GLMP and LAMP1 (included as a negative control) were detected by immunoblot with an antibody against HA. LIMP2 was detected as an additional control and was exclusively detected in the lysates, but not the GFP-precipitate. ( C ) Co-immunoprecipitation of C-terminal GFP-tagged MFSD1 LL/AA PM mutant and HA-tagged GLMP Y400A from transfected HeLa cell lysates solubilized with CHAPS. MFSD1 LL/AA -GFP was precipitated using an antibody against GFP or an unspecific control antibody (cAb). HA-tagged GLMP Y400A and LAMP1 Y414A (PM localized LAMP1 variant included as a negative control) were detected by immunoblot with an antibody against HA. ( D ) Plot of the GFP intensity of the HeLa cells transfected with different plasmids used for the flow cytometry-based FRET analysis. The number in the plot represents the percentage of GFP+ cells alive for each condition.

    Techniques Used: Immunoprecipitation, FACS, Transfection, Negative Control, Mutagenesis, Variant Assay, Flow Cytometry

    39) Product Images from "Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression"

    Article Title: Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0447-9

    Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area
    Figure Legend Snippet: Interaction between HIV-1 Tat and RelB. a , b Myc-Tat (3 μg) was transfected into HEK 293T cells (4 × 10 6 ) together with empty vectors (control) (3 μg) or pFlag-RelB (3 μg) Co-immunoprecipitation was performed with anti-Flag ( a ) or anti-Myc ( b ) antibodies. Samples of both cell lysates and immunoprecipitates were subjected to western blotting and probed with rabbit anti-Myc and anti-Flag antibodies. c Co-IP of endogenous RelB and ectopically expressed Tat. The lysate from HA-Tat-expressing HeLa cells (4 × 10 6 ) was immunoprecipitated with mouse anti-HA antibodies, and the precipitated proteins were examined with western blotting. d Effect of RNases on the association of endogenous RelB and ectopically expressed Tat. Lysates (in the presence or absence of RNase A [5 μg/ml]) of HA-Tat expressing HeLa cells (4 × 10 6 ) were immunoprecipitated with control rabbit IgG or rabbit anti-RelB antibodies. Samples from cell lysates and immunoprecipitates were subjected to western blotting. e Tat partially co-localizes with RelB. HeLa cells (0.1 × 10 6 ) were transfected with HA-Tat (200 ng) and Flag-RelB (200 ng) plasmid DNA. Indirect IFA was performed to detect HA-Tat (with rabbit anti-HA antibody and TRITC-conjugated goat anti rabbit secondary antibody) and Flag-RelB (with mouse anti-Flag antibody and FITC-conjugated goat anti mouse secondary antibody). Nuclei were visualized with DAPI staining. Representative images are shown. The inset shows a higher magnification of the boxed area

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

    40) Product Images from "The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses"

    Article Title: The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erv001

    CaPIK1 interacts with CaChitIV in yeast and in planta. (A) Interaction of CaPIK1 with CaChitIV in a GAL4-based yeast two-hybrid system. Negative control: combination of human lamin C (BD/Lam) and SV40 large T antigen (AD/SV40-T) fusion constructs. Positive control: combination of murine p53 (BD/p53) and SV40 large T antigen (AD/SV40-T) fusion constructs. BD, GAL4 DNA-binding domain; AD, GAL4 activation domain; SD-LT, synthetic dropout medium lacking leucine (L) and tryptophan (T); SD-AHLT X-α-gal, synthetic dropout medium lacking adenine (A), histidine (H), leucine (L), and tryptophan (T) supplemented with X-α-Gal to monitor reporter gene expression. (B) Bimolecular fluorescent complementation (BiFC) analysis of the CaPIK1–CaChitIV interaction in Nicotiana benthamiana . Confocal images were taken from leaf epidermal cells; bZIP63 -YFP N and bZIP63 -YFP C constructs were used as positive controls. Scale bars=50 μm. (C) Co-immunoprecipitation (Co-IP) and immunoblot (IB) analyses of CaChitIV-cMyc and CaPIK1-HA co-expressed in N. benthamiana leaves. Protein loading is shown by Coomassie brilliant blue (CBB) staining. α-cMyc, cMyc antibody; α-HA, HA antibody. (This figure is available in colour at JXB online.)
    Figure Legend Snippet: CaPIK1 interacts with CaChitIV in yeast and in planta. (A) Interaction of CaPIK1 with CaChitIV in a GAL4-based yeast two-hybrid system. Negative control: combination of human lamin C (BD/Lam) and SV40 large T antigen (AD/SV40-T) fusion constructs. Positive control: combination of murine p53 (BD/p53) and SV40 large T antigen (AD/SV40-T) fusion constructs. BD, GAL4 DNA-binding domain; AD, GAL4 activation domain; SD-LT, synthetic dropout medium lacking leucine (L) and tryptophan (T); SD-AHLT X-α-gal, synthetic dropout medium lacking adenine (A), histidine (H), leucine (L), and tryptophan (T) supplemented with X-α-Gal to monitor reporter gene expression. (B) Bimolecular fluorescent complementation (BiFC) analysis of the CaPIK1–CaChitIV interaction in Nicotiana benthamiana . Confocal images were taken from leaf epidermal cells; bZIP63 -YFP N and bZIP63 -YFP C constructs were used as positive controls. Scale bars=50 μm. (C) Co-immunoprecipitation (Co-IP) and immunoblot (IB) analyses of CaChitIV-cMyc and CaPIK1-HA co-expressed in N. benthamiana leaves. Protein loading is shown by Coomassie brilliant blue (CBB) staining. α-cMyc, cMyc antibody; α-HA, HA antibody. (This figure is available in colour at JXB online.)

    Techniques Used: Negative Control, Laser Capture Microdissection, Construct, Positive Control, Binding Assay, Activation Assay, Expressing, Bimolecular Fluorescence Complementation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Staining

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    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of <t>immunoprecipitation</t> eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.
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    86
    Roche immunoprecipitation lysis buffer
    MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in <t>immunoprecipitation</t> lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).
    Immunoprecipitation Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche ice cold immunoprecipitation buffer
    FUS does not bind VAPB or PTPIP51 in <t>immunoprecipitation</t> assays from transfected HEK293 cells Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, myc‐VAPB + CTRL, or myc‐VAPB + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. VAPB was immunoprecipitated via the myc‐tag and the samples probed on immunoblots for VAPB using rabbit VAPB antibody and for co‐immunoprecipitating FUS via the HA tag. Input VAPB and FUS were detected using myc and HA antibodies. Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, HA‐PTPIP51 + CTRL or HA‐PTPIP51 + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. PTPIP51 was immunoprecipitated using rat anti‐PTPIP51 and the samples probed for PTPIP51 using rabbit anti‐HA antibody and for co‐immunoprecipitating FUS using rabbit FUS antibody. Input PTPIP51 and FUS were detected using PTPIP51 and EGFP antibodies, respectively.
    Ice Cold Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche immunoprecipitation ip buffer
    ERβ1 binds to promoters of mutant p53 target genes ( A ) Chromatin <t>immunoprecipitation</t> (ChIP) analysis in control and ERβ1-expressing MDA-MB-231 cells for the presence of ERβ1 at sites of mutant p53 target genes that contain both ERE and p53REs or exclusively ERE ( Follistatin , FST ). ERE/p53REs-negative sites from 36B4 promoter and downstream of the ADAMTS9 gene and a 5′ p53RE from p21 promoter that binds wild-type p53 were used as controls. Anti-FLAG antibody was used to immunoprecipitate ERβ1 and normal mouse IgG was used as experimental control. Fold enrichment of p53 target sequences was normalized to IgG ChIP. ( B ) ChIP analysis for binding of mutant p53 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells. ( C ) ChIP analysis for the presence of ERβ1 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells following transfection with control or p63 siRNA (#1). Fold enrichment of target sequences in ERβ1 precipitates was normalized to that of IgG precipitates. All graphs represent the mean ± SEM of three experiments; * P ≤ 0.05.
    Immunoprecipitation Ip Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Journal: The Journal of Neuroscience

    Article Title: Neuronal Pentraxin 1 Negatively Regulates Excitatory Synapse Density and Synaptic Plasticity

    doi: 10.1523/JNEUROSCI.2548-14.2015

    Figure Lengend Snippet: NP1 interacts and colocalizes with Kv7.2 at presynaptic terminals of excitatory synapses and axonal growth cones. A – D , Representative Western blots of immunoprecipitation eluates separated in 6% Tris-Glycine (for high molecular weight proteins) and 4–12% Bis-Tris gels (for low molecular weight proteins). TL, Total protein lysate; Syx, syntaxin. A , Both native Kv7.2 and syntaxin 1A, but not Kv7.3, coprecipitate with NP1 in total brain extracts. B , Both native Kv7.2 and NP1 coprecipitate with syntaxin in total brain extracts. C , D , Recombinant NP1 coprecipitates Kv7.2, but not syntaxin or Kv7.3, in 293T cells transfected with NP1, 5Myc-Kv7.2, 2HA-Kv7.3, and syntaxin 1A cDNAs. Kv7.2 and Kv7.3 were immunoprecipitated with antibodies against their respective Myc and HA tags. E , F , Immunofluorescence studies and confocal microscopy were performed in high-density ( E ) or low-density ( F ) isolated cortical neurons. E , Top, Confocal sections of 0.772 μm in the z -plane showing immunofluorescence of NP1, VGLUT1, Kv7.2, and negative control (omitting primary antibodies). Bottom, Colocalization (in white) of the excitatory presynaptic marker VGLUT1 (blue) with NP1 (green) and Kv7.2 (red) is shown in a single section with the corresponding orthogonal views of the stack of confocal sections. White arrows indicate sites of colocalization. F , NP1 (green) and KV7.2 (magenta) immunofluorescence and DIC images of an isolated cortical cultured neuron (1×) with its corresponding axonal growth cone highlighted in a white square box, shown in a confocal section of 0.772 μm in the z -plane at higher (5×) magnification. The negative control for primary antibodies is shown in another growth cone on the right. The image in the bottom is the merge of NP1 and Kv7.2 immunofluorescence images in the single confocal section obtained at 5× showing colocalization (white) of NP1 and Kv7.2 in the growth cone, with the corresponding orthogonal views from its respective stack of confocal sections. Images were acquired using restricted spectral emission wavelength ranges chosen to avoid crosstalk or bleed-through between the three different channels. Scale bar, 5 μm.

    Article Snippet: For total rat brain extraction, the whole rat brain was homogenized in 10 volumes of immunoprecipitation buffer (50 m m Tris-HCl, pH 7.5, 100 m m NaCl, 2 m m CaCl2 , 1% Triton X-100) containing the mini-EDTA-free protease inhibitor cocktail (Roche).

    Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Recombinant, Transfection, Immunofluorescence, Confocal Microscopy, Isolation, Negative Control, Marker, Cell Culture

    MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in immunoprecipitation lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).

    Journal: The Journal of Biological Chemistry

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

    doi: 10.1074/jbc.M113.468579

    Figure Lengend Snippet: MAGE-A11 stabilizes E2F1 and mediates an interaction between E2F1 and p107. A , pCMV5 (1 μg) ( lane 1 ), 1 μg of pSG5-MAGE ( lanes 2 and 3 ), 0.5 μg of CMV-E2F1 ( lanes 4 and 5 ), or 1 μg of pSG5-MAGE and 0.5 μg of CMV-E2F1 together ( lanes 6 and 7 ) was expressed in COS1 cells. Cells were incubated for 24 h before harvest in serum-free medium with or without 0.1 μg/ml EGF. Cell extracts (40 μg of protein/lane) were probed on the transblot using E2F1 (sc-193) and MAGE-A11 antibodies. B , pCMV-FLAG (4 μg) (−) or 4 μg of pCMV-FLAG-p107 was expressed in COS1 cells with or without 3 μg of pSG5-MAGE or 4 μg of CMV-E2F1 alone or together. Cells were incubated for 24 h in serum-free medium with 0.1 μg/ml EGF and immunoprecipitated using FLAG affinity resin. Transblots of cell extracts (40 μg of protein/lane, left ) or immunoprecipitates ( IP , right ) were probed using p107, E2F1 (sc-193), and MAGE-A11 antibodies. C , pCMV-FLAG, or pCMV-FLAG-p107 (4 μg/dish) was expressed in three 10-cm dishes plated at 7 × 10 6 LAPC-4 cells/dish using 8 μl of X-tremeGENE in 160 μl of medium added/dish. The next day, cells were transferred to serum-free medium with 0.1 μg/ml EGF and incubated for 24 h. The cell extract (40 μg of protein/lane) prepared in immunoprecipitation lysis buffer and immunoprecipitates were probed overnight at 4 °C on transblots using p107 (sc-318, 1:200 dilution) and MAGE-A11 antibodies (15 μg/ml). The blot was stripped and reprobed using E2F1 (sc-193) antibody (1:100 dilution).

    Article Snippet: Cell pellets were extracted in immunoprecipitation lysis buffer containing 1% Triton X-100, 0.15 m NaCl, 50 m m NaF, 2 m m sodium vanadate, 2 m m EDTA, 50 m m Tris (pH 7.6), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and complete protease inhibitors (Roche Applied Science) with or without 0.5% deoxycholate or 10% glycerol.

    Techniques: Incubation, Immunoprecipitation, Lysis

    MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 ( A ), pCMV-hRb ( B ), or pcDNA3-p130 ( C ). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right ) and immunoprecipitates ( IP , left ) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows , p107, Rb, p130, FLAG-MAGE, and IgG.

    Journal: The Journal of Biological Chemistry

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *

    doi: 10.1074/jbc.M113.468579

    Figure Lengend Snippet: MAGE-A11 interacts with p107 and Rb but not p130. pCMV-FLAG (−), pCMV-FLAG-MAGE, or FLAG-MAGE-(112–429) (5 μg) was expressed in COS1 cells with 5 μg of CMV-p107 ( A ), pCMV-hRb ( B ), or pcDNA3-p130 ( C ). Cells were incubated for 24 h in serum-free medium containing 0.1 μg/ml EGF and harvested in immunoprecipitation lysis buffer without deoxycholate or glycerol. Cell extracts (50 μg of protein/lane, right ) and immunoprecipitates ( IP , left ) were probed using p107, Rb, p130, and MAGE-A11 antibodies. Arrows , p107, Rb, p130, FLAG-MAGE, and IgG.

    Article Snippet: Cell pellets were extracted in immunoprecipitation lysis buffer containing 1% Triton X-100, 0.15 m NaCl, 50 m m NaF, 2 m m sodium vanadate, 2 m m EDTA, 50 m m Tris (pH 7.6), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and complete protease inhibitors (Roche Applied Science) with or without 0.5% deoxycholate or 10% glycerol.

    Techniques: Incubation, Immunoprecipitation, Lysis

    FUS does not bind VAPB or PTPIP51 in immunoprecipitation assays from transfected HEK293 cells Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, myc‐VAPB + CTRL, or myc‐VAPB + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. VAPB was immunoprecipitated via the myc‐tag and the samples probed on immunoblots for VAPB using rabbit VAPB antibody and for co‐immunoprecipitating FUS via the HA tag. Input VAPB and FUS were detected using myc and HA antibodies. Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, HA‐PTPIP51 + CTRL or HA‐PTPIP51 + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. PTPIP51 was immunoprecipitated using rat anti‐PTPIP51 and the samples probed for PTPIP51 using rabbit anti‐HA antibody and for co‐immunoprecipitating FUS using rabbit FUS antibody. Input PTPIP51 and FUS were detected using PTPIP51 and EGFP antibodies, respectively.

    Journal: EMBO Reports

    Article Title: ALS/ FTD‐associated FUS activates GSK‐3β to disrupt the VAPB– PTPIP51 interaction and ER–mitochondria associations

    doi: 10.15252/embr.201541726

    Figure Lengend Snippet: FUS does not bind VAPB or PTPIP51 in immunoprecipitation assays from transfected HEK293 cells Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, myc‐VAPB + CTRL, or myc‐VAPB + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. VAPB was immunoprecipitated via the myc‐tag and the samples probed on immunoblots for VAPB using rabbit VAPB antibody and for co‐immunoprecipitating FUS via the HA tag. Input VAPB and FUS were detected using myc and HA antibodies. Cells were transfected as indicated with control vector (CTRL), HA‐FUS + CTRL, HA‐PTPIP51 + CTRL or HA‐PTPIP51 + either HA‐FUS, HA‐FUSR521C or HA‐FUSR518K. PTPIP51 was immunoprecipitated using rat anti‐PTPIP51 and the samples probed for PTPIP51 using rabbit anti‐HA antibody and for co‐immunoprecipitating FUS using rabbit FUS antibody. Input PTPIP51 and FUS were detected using PTPIP51 and EGFP antibodies, respectively.

    Article Snippet: Immunoprecipitations, SDS–PAGE and immunoblotting Cells were harvested for SDS–PAGE and immunoblotting by scraping into SDS–PAGE sample buffer containing 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.1% bromophenol blue and protease inhibitors (Complete Roche) in 50 mM Tris–HCl pH 6.8 and heating to 100°C for 5 min. Transgenic mouse samples were homogenized in 50 mM Tris–citrate pH 7.4, 150 mM NaCl, 1% Triton X‐100, 5 mM ethylene glycol tetraacetic acid, 5 mM EDTA and protease inhibitors (Complete, Roche) and then prepared for SDS–PAGE by addition of SDS–PAGE sample buffer and heating to 100°C for 5 min. For immunoprecipitation assays, transfected cells were lysed in ice‐cold immunoprecipitation buffer comprising 50 mM Tris–citrate pH 7.4, 150 mM NaCl, 1% Triton X‐100, 5 mM ethylene glycol tetraacetic acid, 5 mM EDTA and protease inhibitors (Complete, Roche).

    Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot

    ERβ1 binds to promoters of mutant p53 target genes ( A ) Chromatin immunoprecipitation (ChIP) analysis in control and ERβ1-expressing MDA-MB-231 cells for the presence of ERβ1 at sites of mutant p53 target genes that contain both ERE and p53REs or exclusively ERE ( Follistatin , FST ). ERE/p53REs-negative sites from 36B4 promoter and downstream of the ADAMTS9 gene and a 5′ p53RE from p21 promoter that binds wild-type p53 were used as controls. Anti-FLAG antibody was used to immunoprecipitate ERβ1 and normal mouse IgG was used as experimental control. Fold enrichment of p53 target sequences was normalized to IgG ChIP. ( B ) ChIP analysis for binding of mutant p53 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells. ( C ) ChIP analysis for the presence of ERβ1 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells following transfection with control or p63 siRNA (#1). Fold enrichment of target sequences in ERβ1 precipitates was normalized to that of IgG precipitates. All graphs represent the mean ± SEM of three experiments; * P ≤ 0.05.

    Journal: Oncotarget

    Article Title: ERβ decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function

    doi: 10.18632/oncotarget.7300

    Figure Lengend Snippet: ERβ1 binds to promoters of mutant p53 target genes ( A ) Chromatin immunoprecipitation (ChIP) analysis in control and ERβ1-expressing MDA-MB-231 cells for the presence of ERβ1 at sites of mutant p53 target genes that contain both ERE and p53REs or exclusively ERE ( Follistatin , FST ). ERE/p53REs-negative sites from 36B4 promoter and downstream of the ADAMTS9 gene and a 5′ p53RE from p21 promoter that binds wild-type p53 were used as controls. Anti-FLAG antibody was used to immunoprecipitate ERβ1 and normal mouse IgG was used as experimental control. Fold enrichment of p53 target sequences was normalized to IgG ChIP. ( B ) ChIP analysis for binding of mutant p53 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells. ( C ) ChIP analysis for the presence of ERβ1 at sites of mutant p53 target genes in control and ERβ1-expressing MDA-MB-231 cells following transfection with control or p63 siRNA (#1). Fold enrichment of target sequences in ERβ1 precipitates was normalized to that of IgG precipitates. All graphs represent the mean ± SEM of three experiments; * P ≤ 0.05.

    Article Snippet: Co-immunoprecipitation and immunoblotting Cells were plated at a density of 5 × 105 −106 per 10-cm dish and lysed in immunoprecipitation (IP) buffer containing 50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.8 mM EGTA, 1 mM NP-40, 1 mM Glycerol, 2 mM PMSF, 1 mM Na3 V04 , 50 mM NaF, 1% protease inhibitor cocktail (Roche) and 1% phosphatase inhibitor mixture (Sigma).

    Techniques: Mutagenesis, Chromatin Immunoprecipitation, Expressing, Multiple Displacement Amplification, Binding Assay, Transfection