immunoprecipitations  (Roche)


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    Structured Review

    Roche immunoprecipitations
    Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line <t>immunoprecipitations</t> by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and
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    Images

    1) Product Images from "Characterization of early autophagy signaling by quantitative phosphoproteomics"

    Article Title: Characterization of early autophagy signaling by quantitative phosphoproteomics

    Journal: Autophagy

    doi: 10.4161/auto.26864

    Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line immunoprecipitations by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and
    Figure Legend Snippet: Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line immunoprecipitations by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and

    Techniques Used:

    2) Product Images from "Fidelity of G protein ?-subunit association by the G protein ?-subunit-like domains of RGS6, RGS7, and RGS11"

    Article Title: Fidelity of G protein ?-subunit association by the G protein ?-subunit-like domains of RGS6, RGS7, and RGS11

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

    doi:

    In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.
    Figure Legend Snippet: In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

    Techniques Used: In Vitro, Binding Assay, Sequencing, Mutagenesis, Immunoprecipitation, SDS Page, Autoradiography

    3) Product Images from "The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch ActivationProtein's "Part-Time Job" Reveals New Facet of Signaling Pathway"

    Article Title: The Translation Initiation Factor 3f (eIF3f) Exhibits a Deubiquitinase Activity Regulating Notch ActivationProtein's "Part-Time Job" Reveals New Facet of Signaling Pathway

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000545

    Interaction of eIF3f and activated Notch in the presence of DTX. HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.
    Figure Legend Snippet: Interaction of eIF3f and activated Notch in the presence of DTX. HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.

    Techniques Used: Construct, Western Blot

    Interactions of eIF3f and DTX. (A) Schematic representation of murine eIF3f deletion mutants. The conserved MPN domain is depicted in blue; amino acid coordinates are indicated. (B) HEK293T cells were cotransfected with vectors encoding HA-tagged eIF3f WT or (1–192), and VSV-tagged DTX. Whole cell extracts (WCE, 5% of the lysates) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-HA antibody. β-tubulin was used as a loading control. (C) HEK293T cells were cotransfected with vectors encoding Flag-tagged eIF3f (188–361) or (91–361), and VSV-tagged DTX. WCE (5%) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-Flag antibody. β-tubulin was used as a loading control. (D) HEK293T cells were cotransfected with vectors encoding the various eIF3f mutants and VSV-DTX. WCE were immunoprecipitated with anti-VSV antibody, and the precipitates were eluted with VSV peptide before being loaded on SDS gels. The eluted material and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. (E) Stable cell lines derived form MEFs by retroviral transduction of VSV-DTX or S-tagged eIF3f (either WT or active site mutant) were lysed and subjected to parallel immunoprecipitations with VSV and S-tag antibodies as indicated. The VSV- or Laemmli-eluted material (for VSV and S-tag IPs, respectively) and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. White lines indicate that intervening lanes have been spliced out.
    Figure Legend Snippet: Interactions of eIF3f and DTX. (A) Schematic representation of murine eIF3f deletion mutants. The conserved MPN domain is depicted in blue; amino acid coordinates are indicated. (B) HEK293T cells were cotransfected with vectors encoding HA-tagged eIF3f WT or (1–192), and VSV-tagged DTX. Whole cell extracts (WCE, 5% of the lysates) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-HA antibody. β-tubulin was used as a loading control. (C) HEK293T cells were cotransfected with vectors encoding Flag-tagged eIF3f (188–361) or (91–361), and VSV-tagged DTX. WCE (5%) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-Flag antibody. β-tubulin was used as a loading control. (D) HEK293T cells were cotransfected with vectors encoding the various eIF3f mutants and VSV-DTX. WCE were immunoprecipitated with anti-VSV antibody, and the precipitates were eluted with VSV peptide before being loaded on SDS gels. The eluted material and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. (E) Stable cell lines derived form MEFs by retroviral transduction of VSV-DTX or S-tagged eIF3f (either WT or active site mutant) were lysed and subjected to parallel immunoprecipitations with VSV and S-tag antibodies as indicated. The VSV- or Laemmli-eluted material (for VSV and S-tag IPs, respectively) and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. White lines indicate that intervening lanes have been spliced out.

    Techniques Used: Immunoprecipitation, Western Blot, Stable Transfection, Derivative Assay, Transduction, Mutagenesis

    4) Product Images from "CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage"

    Article Title: CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks222

    DNA damage-induced sumoylation of BMI1 at lysine 88. ( A ) 293T cells transiently transfected with CBX4, myc-tagged BMI1, Ubc9 and YFP-SUMO-1 were irradiated with 6 Gy and permitted to recover for 1 h at 37°C. Nuclear extracts were prepared and immunoprecipitated using myc or GFP-specific antibody. Immunoblot analysis was done using BMI1 antibody. As a control different blots were run simultaneously and probed with γ-H2AX and actin. ( B ) BMI1 KO MEFs reconstituted either with empty vector, GFP-BMI1 WT or GFP-BMI1 K88R constructs were treated as in (A). Nuclear extracts and immunoprecipitations were done using GFP antibody. Immunoblot analysis was done using BMI1 antibody. ( C ) Time-lapse fluorescence microscopy of GFP- WT BMI1, GFP- K88R-BMI1, GFP- WT-BMI1-SUMO1 or GFP-K88R-BMI1-SUMO-1 in U2OS following laser micro-irradiation. The fluorescence intensity values in the micro-irradiated areas were pooled from 10 to 15 independent cells from three independent experiments and plotted on a time scale. ( D ) U2OS cells expressing Ubc9 and SUMO-1 were transfected with either control or one of the two different CBX4, shRNA were irradiated with 6 Gy and permitted to recover for 1 h at 37°C. Nuclear extracts were immunoprecipitated and immunoblotted using BMI1 antibody. ( E ) CBX4 promotes SUMO-1 conjugation to BMI1 in vitro . Purified proteins GST-tagged BMI1 and GST-tagged CBX4 proteins were incubated with recombinant Aos1/Uba2 (E1), Ubc9 and SUMO-1 as indicated. The reaction mixture was analyzed by western blotting using anti-BMI1antibody.
    Figure Legend Snippet: DNA damage-induced sumoylation of BMI1 at lysine 88. ( A ) 293T cells transiently transfected with CBX4, myc-tagged BMI1, Ubc9 and YFP-SUMO-1 were irradiated with 6 Gy and permitted to recover for 1 h at 37°C. Nuclear extracts were prepared and immunoprecipitated using myc or GFP-specific antibody. Immunoblot analysis was done using BMI1 antibody. As a control different blots were run simultaneously and probed with γ-H2AX and actin. ( B ) BMI1 KO MEFs reconstituted either with empty vector, GFP-BMI1 WT or GFP-BMI1 K88R constructs were treated as in (A). Nuclear extracts and immunoprecipitations were done using GFP antibody. Immunoblot analysis was done using BMI1 antibody. ( C ) Time-lapse fluorescence microscopy of GFP- WT BMI1, GFP- K88R-BMI1, GFP- WT-BMI1-SUMO1 or GFP-K88R-BMI1-SUMO-1 in U2OS following laser micro-irradiation. The fluorescence intensity values in the micro-irradiated areas were pooled from 10 to 15 independent cells from three independent experiments and plotted on a time scale. ( D ) U2OS cells expressing Ubc9 and SUMO-1 were transfected with either control or one of the two different CBX4, shRNA were irradiated with 6 Gy and permitted to recover for 1 h at 37°C. Nuclear extracts were immunoprecipitated and immunoblotted using BMI1 antibody. ( E ) CBX4 promotes SUMO-1 conjugation to BMI1 in vitro . Purified proteins GST-tagged BMI1 and GST-tagged CBX4 proteins were incubated with recombinant Aos1/Uba2 (E1), Ubc9 and SUMO-1 as indicated. The reaction mixture was analyzed by western blotting using anti-BMI1antibody.

    Techniques Used: Transfection, Irradiation, Immunoprecipitation, Plasmid Preparation, Construct, Fluorescence, Microscopy, Expressing, shRNA, Conjugation Assay, In Vitro, Purification, Incubation, Recombinant, Western Blot

    5) Product Images from "Leveraging New Definitions of the LxVP SLiM To Discover Novel Calcineurin Regulators and Substrates"

    Article Title: Leveraging New Definitions of the LxVP SLiM To Discover Novel Calcineurin Regulators and Substrates

    Journal: ACS chemical biology

    doi: 10.1021/acschembio.9b00606

    The SBSC-MS analysis of CN. (A) Structure of CN (A: catalytic domain, black; B: regulatory domain, cyan) bound to the AKAP PxIxIT SLiM (green, PDBID: 3LL8) within the PxIxIT binding pocket (dark blue) and the NFATc1 LxVP SLiM (magenta, PDBID: 5SVE) bound within the LxVP binding pocket (light blue). CN active site metals Zn 2+ and Fe 3+ : yellow and orange spheres, respectively. CNB Ca 2+ ions: red spheres. (B) Close-up of the NFATc1 LxVP bound to the CN LxVP binding pocket (blue). Binding pocket core (dashed outline) with highlighted areas (yellow) shows the interaction surfaces for the i, i+2, and i+3 residues in the LxVP motif. (C) Experimental setup to identify CNA interactors. (D) Volcano plot of label-free AP-MS identification of CN interactors. Red: known CN interactors; blue: new interactors selected for validation. (E) FLAG-IP schematic for validation of CNA interactions. (F) Western blot analysis of immunoprecipitations from nontransfected control cells and cells expressing FLAG-tagged protein and their interaction with CNA.
    Figure Legend Snippet: The SBSC-MS analysis of CN. (A) Structure of CN (A: catalytic domain, black; B: regulatory domain, cyan) bound to the AKAP PxIxIT SLiM (green, PDBID: 3LL8) within the PxIxIT binding pocket (dark blue) and the NFATc1 LxVP SLiM (magenta, PDBID: 5SVE) bound within the LxVP binding pocket (light blue). CN active site metals Zn 2+ and Fe 3+ : yellow and orange spheres, respectively. CNB Ca 2+ ions: red spheres. (B) Close-up of the NFATc1 LxVP bound to the CN LxVP binding pocket (blue). Binding pocket core (dashed outline) with highlighted areas (yellow) shows the interaction surfaces for the i, i+2, and i+3 residues in the LxVP motif. (C) Experimental setup to identify CNA interactors. (D) Volcano plot of label-free AP-MS identification of CN interactors. Red: known CN interactors; blue: new interactors selected for validation. (E) FLAG-IP schematic for validation of CNA interactions. (F) Western blot analysis of immunoprecipitations from nontransfected control cells and cells expressing FLAG-tagged protein and their interaction with CNA.

    Techniques Used: Binding Assay, Western Blot, Expressing

    6) Product Images from "Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake"

    Article Title: Anthrax toxin triggers the activation of src-like kinases to mediate its own uptake

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

    doi: 10.1073/pnas.0910782107

    Phosphorylationof CMG2 is required for its ubiquitination. HeLa cells were transfected for 48 h with CMG2-WT-HA or CMG2 ∆Y -HA mutated on four tyrosines: Y380A, Y381A, Y445A, and Y463A. Cells were then treated with 500 g/mL of PA83 for indicated times at 37 °C. Immunoprecipitations were performed against HA, and the samples were analyzed by Western blotting both against the HA-tagged receptor and against ubiquitin.
    Figure Legend Snippet: Phosphorylationof CMG2 is required for its ubiquitination. HeLa cells were transfected for 48 h with CMG2-WT-HA or CMG2 ∆Y -HA mutated on four tyrosines: Y380A, Y381A, Y445A, and Y463A. Cells were then treated with 500 g/mL of PA83 for indicated times at 37 °C. Immunoprecipitations were performed against HA, and the samples were analyzed by Western blotting both against the HA-tagged receptor and against ubiquitin.

    Techniques Used: Transfection, Western Blot

    7) Product Images from "The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3? Processing Factors ▿The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3? Processing Factors ▿ †"

    Article Title: The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3? Processing Factors ▿The Rpb4 Subunit of RNA Polymerase II Contributes to Cotranscriptional Recruitment of 3? Processing Factors ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01714-07

    Association of Rna14 with RNApII is dependent on Rpb4. IgG-agarose was used to precipitate TAP-tagged Rna14 from whole-cell extracts of wild-type (YSB1147) and rpb4 Δ (YSB2033) strains. An untagged wild-type strain (YSB1140) was also included as a control. Immunoprecipitations were done both in the absence (−) and in the presence (+) of RNase A. Immunoblot analysis was then performed using 8WG16 antibody against Rpb1 (top) as well as the PAP antibody to detect expression of TAP-tagged Rna14 (bottom).
    Figure Legend Snippet: Association of Rna14 with RNApII is dependent on Rpb4. IgG-agarose was used to precipitate TAP-tagged Rna14 from whole-cell extracts of wild-type (YSB1147) and rpb4 Δ (YSB2033) strains. An untagged wild-type strain (YSB1140) was also included as a control. Immunoprecipitations were done both in the absence (−) and in the presence (+) of RNase A. Immunoblot analysis was then performed using 8WG16 antibody against Rpb1 (top) as well as the PAP antibody to detect expression of TAP-tagged Rna14 (bottom).

    Techniques Used: Expressing

    8) Product Images from "UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument"

    Article Title: UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument

    Journal: Journal of Virology

    doi: 10.1128/JVI.00474-20

    The composition of the tegument layer is altered in virions produced in the absence of UL88. (A) Western blot analysis of WT TB40/E, UL88-STOP, and UL88-REV purified on a sodium tartrate gradient. The blots were probed for the envelope protein gB and the tegument proteins UL88, UL47, UL48, pp71, pp150, UL71, pp28, and pp65. Gradient purifications were done for three distinct preparations of each virus, and the blots shown represent one of these preparations. (B) Western blot analysis of fibroblasts infected (MOI, 3) with WT TB40/E, UL88-STOP, UL88- galK , and UL88-REV at 96 hpi. The blots were probed with antibodies against the tegument proteins UL88, UL47, UL48, pp150, pp28, and pp65 and tubulin as a loading control. Results of quantitative analysis of the blots in panels A and B plus two additional repeats are shown to the right of the blots. Error bars represent standard deviations. (C) Quantitative PCR analysis of UL48 transcript levels at 96 hpi in fibroblasts infected with WT TB40/E, UL88-STOP, and UL88-REV. Values are relative to the wild-type sample after normalization and are averages for three biological replicates. (D) Western blot analysis of immunoprecipitations using FLAG beads of HEK293TN cell lysates prepared 24 h after cotransfection with UL88 and either UL47-FLAG or vector control. Blots were probed for FLAG and UL88. (E) Fibroblasts expressing a vector control or UL47-FLAG were infected with WT TB40/E (MOI, 3), and lysates were subjected to immunoprecipitation analysis with FLAG beads at 96 hpi. Blots were probed for antibodies against FLAG, UL88, and tubulin. *, IgG heavy chain; ‡, nonspecific band.
    Figure Legend Snippet: The composition of the tegument layer is altered in virions produced in the absence of UL88. (A) Western blot analysis of WT TB40/E, UL88-STOP, and UL88-REV purified on a sodium tartrate gradient. The blots were probed for the envelope protein gB and the tegument proteins UL88, UL47, UL48, pp71, pp150, UL71, pp28, and pp65. Gradient purifications were done for three distinct preparations of each virus, and the blots shown represent one of these preparations. (B) Western blot analysis of fibroblasts infected (MOI, 3) with WT TB40/E, UL88-STOP, UL88- galK , and UL88-REV at 96 hpi. The blots were probed with antibodies against the tegument proteins UL88, UL47, UL48, pp150, pp28, and pp65 and tubulin as a loading control. Results of quantitative analysis of the blots in panels A and B plus two additional repeats are shown to the right of the blots. Error bars represent standard deviations. (C) Quantitative PCR analysis of UL48 transcript levels at 96 hpi in fibroblasts infected with WT TB40/E, UL88-STOP, and UL88-REV. Values are relative to the wild-type sample after normalization and are averages for three biological replicates. (D) Western blot analysis of immunoprecipitations using FLAG beads of HEK293TN cell lysates prepared 24 h after cotransfection with UL88 and either UL47-FLAG or vector control. Blots were probed for FLAG and UL88. (E) Fibroblasts expressing a vector control or UL47-FLAG were infected with WT TB40/E (MOI, 3), and lysates were subjected to immunoprecipitation analysis with FLAG beads at 96 hpi. Blots were probed for antibodies against FLAG, UL88, and tubulin. *, IgG heavy chain; ‡, nonspecific band.

    Techniques Used: Produced, Western Blot, Purification, Infection, Real-time Polymerase Chain Reaction, Cotransfection, Plasmid Preparation, Expressing, Immunoprecipitation

    9) Product Images from "Optineurin Is Required for CYLD-Dependent Inhibition of TNF?-Induced NF-?B Activation"

    Article Title: Optineurin Is Required for CYLD-Dependent Inhibition of TNF?-Induced NF-?B Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017477

    Effect of H486R optineurin on CYLD-dependent inhibition of TNFα-induced NF-κB activity. HeLa cells were transfected with optineurin or its mutants (100 ng) along with or without CYLD (100 ng, left panel and 50 ng, right panel). After 22 h of transfection, the cells were treated with TNFα for 4 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot showing the expression of optineurin and its mutants along with CYLD using HA tag antibody. Yeast strain PJ694A was co-transformed with optineurin or its H486R and D474N mutants and CYLD. Transformants were grown on selection media lacking Ade to assay interaction. Growth on Ade − plate indicates interaction. GST-ubiquitin or GST alone bound to glutathione agarose beads were incubated with lysates of HEK293T cells transfected with wild type optineurin or its mutants. The bound proteins were eluted and immunoblotted with anti-HA antibodies. WCL, whole cell lysates. HeLa cells were infected with adenoviruses expressing HA-tagged wild-type or H486R mutant optineurin. After 30 hrs of infection, the cells were treated with TNFα for 5 min and immunoprecipitations were carried out with HA antibody and analyzed by Western blotting with RIP and HA antibodies.
    Figure Legend Snippet: Effect of H486R optineurin on CYLD-dependent inhibition of TNFα-induced NF-κB activity. HeLa cells were transfected with optineurin or its mutants (100 ng) along with or without CYLD (100 ng, left panel and 50 ng, right panel). After 22 h of transfection, the cells were treated with TNFα for 4 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot showing the expression of optineurin and its mutants along with CYLD using HA tag antibody. Yeast strain PJ694A was co-transformed with optineurin or its H486R and D474N mutants and CYLD. Transformants were grown on selection media lacking Ade to assay interaction. Growth on Ade − plate indicates interaction. GST-ubiquitin or GST alone bound to glutathione agarose beads were incubated with lysates of HEK293T cells transfected with wild type optineurin or its mutants. The bound proteins were eluted and immunoblotted with anti-HA antibodies. WCL, whole cell lysates. HeLa cells were infected with adenoviruses expressing HA-tagged wild-type or H486R mutant optineurin. After 30 hrs of infection, the cells were treated with TNFα for 5 min and immunoprecipitations were carried out with HA antibody and analyzed by Western blotting with RIP and HA antibodies.

    Techniques Used: Inhibition, Activity Assay, Transfection, Luciferase, Western Blot, Expressing, Transformation Assay, Selection, Incubation, Infection, Mutagenesis

    10) Product Images from "The RNA Helicase DHX34 Activates NMD by Promoting a Transition from the Surveillance to the Decay-Inducing Complex"

    Article Title: The RNA Helicase DHX34 Activates NMD by Promoting a Transition from the Surveillance to the Decay-Inducing Complex

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2014.08.020

    DHX34 Promotes eRF3 Release from the SURF Complex (A) FLAG-UPF1 was transiently coexpressed with T7-DHX34 in HEK293T cells. Inputs (0.5%) and anti-FLAG IPs (20%) were analyzed with the indicated antibodies. (B) HEK293T cells were transiently transfected with FLAG-UPF1 and with an shRNA targeting DHX34 or with an empty vector control. Inputs (0.5%) and anti-FLAG (20%) IPs were analyzed by western blotting for the indicated proteins. (C and D) Cells were transfected with an shRNA targeting DHX34 or with an empty vector control. After 4 days cells were cotransfected with FLAG-eRF1 (C) or FLAG-eRF3 (D) and shRNA-resistant ( R ) T7-DHX34 (wild-type or the ATPase-deficient mutants K191S and D279A). Anti-FLAG immunoprecipitations were performed 2 days later and analyzed for the presence of DHX34. Input (0.5%) and anti-FLAG IPs (20%) were analyzed by western blotting with the indicated antibodies.
    Figure Legend Snippet: DHX34 Promotes eRF3 Release from the SURF Complex (A) FLAG-UPF1 was transiently coexpressed with T7-DHX34 in HEK293T cells. Inputs (0.5%) and anti-FLAG IPs (20%) were analyzed with the indicated antibodies. (B) HEK293T cells were transiently transfected with FLAG-UPF1 and with an shRNA targeting DHX34 or with an empty vector control. Inputs (0.5%) and anti-FLAG (20%) IPs were analyzed by western blotting for the indicated proteins. (C and D) Cells were transfected with an shRNA targeting DHX34 or with an empty vector control. After 4 days cells were cotransfected with FLAG-eRF1 (C) or FLAG-eRF3 (D) and shRNA-resistant ( R ) T7-DHX34 (wild-type or the ATPase-deficient mutants K191S and D279A). Anti-FLAG immunoprecipitations were performed 2 days later and analyzed for the presence of DHX34. Input (0.5%) and anti-FLAG IPs (20%) were analyzed by western blotting with the indicated antibodies.

    Techniques Used: Transfection, shRNA, Plasmid Preparation, Western Blot

    11) Product Images from "Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment"

    Article Title: Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

    Journal: mBio

    doi: 10.1128/mBio.01433-16

    Interactions of HIV-1 Vpr with SLX4 are variable. Vpr from multiple isolates closely related to HVI-1 or HIV-2 were tested for their ability to interact with human SLX4 by coimmunoprecipitation. (A) HIV-1 group M Vpr isolates: HIV-1 LAI (subtype B), Q23-17 (subtype A), SE6165 (subtype G), SE9280 (subtype J), and ETH2220 (subtype C). (B) HIV-2 and related Vpr orthologs. (C) HIV-1 groups and ancestral SIVcpz and SIVgor Vpr orthologs. 3× FLAG Vpr was transiently coexpressed with HA-tagged SLX4 in 293T cells (lower panels), and immunoprecipitations against the HA tag were performed (upper panels). HIV-1 LAI Vpr was used as a positive control. The loading control was whole-cell stain (Input), tubulin (Input), or IgG heavy chain (HA IP). See Fig. S2 in the supplemental material for related data, including longer exposures for FLAG-Vpr following HA-IP.
    Figure Legend Snippet: Interactions of HIV-1 Vpr with SLX4 are variable. Vpr from multiple isolates closely related to HVI-1 or HIV-2 were tested for their ability to interact with human SLX4 by coimmunoprecipitation. (A) HIV-1 group M Vpr isolates: HIV-1 LAI (subtype B), Q23-17 (subtype A), SE6165 (subtype G), SE9280 (subtype J), and ETH2220 (subtype C). (B) HIV-2 and related Vpr orthologs. (C) HIV-1 groups and ancestral SIVcpz and SIVgor Vpr orthologs. 3× FLAG Vpr was transiently coexpressed with HA-tagged SLX4 in 293T cells (lower panels), and immunoprecipitations against the HA tag were performed (upper panels). HIV-1 LAI Vpr was used as a positive control. The loading control was whole-cell stain (Input), tubulin (Input), or IgG heavy chain (HA IP). See Fig. S2 in the supplemental material for related data, including longer exposures for FLAG-Vpr following HA-IP.

    Techniques Used: Positive Control, Staining

    HIV-1 and HIV-2 Vpr differentially engage the host DNA damage response. (A) Representative immunofluorescence images of U2OS cells expressing 3× FLAG-tagged Vpr, control empty vector (no Vpr), or uninfected cells. FANCD2 foci show activation of the DNA damage response. Blue (DAPI) shows the nuclei, 3× FLAG Vpr is shown in red, and FANCD2 is shown in green. Values indicate percentages of FANCD2-positive cells (more than 6 FANCD2 foci) that were also Vpr positive. For negative controls, values are simply the percentages of FANCD2-positive cells in the entire field ( n = > 15). (B) HA-tagged human SLX4 and FLAG-tagged Vpr were transiently coexpressed in 293T cells (left panels), and immunoprecipitations against the HA tag were performed (right panels). The loading control was either whole-cell stain (Input) or IgG heavy chain (HA IP) (note that there is a small amount of sample spillover in the HIV-2 Rod9-negative SLX4 input lane from the adjacent lane). See Fig. S1 in the supplemental material for related data.
    Figure Legend Snippet: HIV-1 and HIV-2 Vpr differentially engage the host DNA damage response. (A) Representative immunofluorescence images of U2OS cells expressing 3× FLAG-tagged Vpr, control empty vector (no Vpr), or uninfected cells. FANCD2 foci show activation of the DNA damage response. Blue (DAPI) shows the nuclei, 3× FLAG Vpr is shown in red, and FANCD2 is shown in green. Values indicate percentages of FANCD2-positive cells (more than 6 FANCD2 foci) that were also Vpr positive. For negative controls, values are simply the percentages of FANCD2-positive cells in the entire field ( n = > 15). (B) HA-tagged human SLX4 and FLAG-tagged Vpr were transiently coexpressed in 293T cells (left panels), and immunoprecipitations against the HA tag were performed (right panels). The loading control was either whole-cell stain (Input) or IgG heavy chain (HA IP) (note that there is a small amount of sample spillover in the HIV-2 Rod9-negative SLX4 input lane from the adjacent lane). See Fig. S1 in the supplemental material for related data.

    Techniques Used: Immunofluorescence, Expressing, Plasmid Preparation, Activation Assay, Staining

    12) Product Images from "Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity) Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity"

    Article Title: Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity) Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity

    Journal: Cellular Microbiology

    doi: 10.1111/cmi.12559

    Phosphorylation of NS1 T49 leads to reduced vRNA, RIG‐I and TRIM25 binding as well as structural destabilization of the RBD. A. A549 cells were transfected with pcDNA3 plasmids containing wt NS1 or NS1 with indicated mutations or were mock transfected. Cell lysates were subjected to immunoprecipitations 24 h p.i. using mouse anti‐NS1 antibody. Washed beads were incubated with vRNA and RNA bound to immunocomplexes was extracted. The relative amount of viral NS1 mRNA was determined by qRT‐PCR as described previously (Habjan et al ., 2008 ). Data represents mean ± SD of two independently repeated experiments. B. Comparative binding scores of RNA to RBD T49E, T49A and T49 as calculated using random forest model from the RNA–protein interaction prediction (RPISeq) tool (Muppirala et al ., 2011 ). The RNA sequence was obtained from Protein Data Bank (PDB) ID 2ZKO (Cheng et al ., 2009 ). C. Structural simulation on 2ZKO structure employing DUET (Pires et al ., 2014b ) was predicted as destabilizing (−0.17 Kcal/mol) resulting in an unfavourable RNA binding ability. The figure shows the stereo‐chemical effect of E49 compared with T49. The RNA helix is shown in yellow, while the two monomers of the NS1 dimer are shown in green and blue. NS1 residues T49 and E49 are shown in red and purple respectively. Molecular graphic simulation was performed using Bioblender (Andrei et al ., 2012 ). D. For analysis of NS1‐RIG‐I interaction, HEK293 cells transiently expressing FLAG‐tagged RIG‐I and the indicated NS1 proteins were subjected to crosslinking with DSP after 48 h, followed by quenching with glycine. Control cells were mock transfected. FLAG‐tagged RIG‐I was immunoprecipitated with anti‐FLAG M2 antibody. Detection of FLAG‐tagged RIG‐I and co‐precipitated NS1 protein was performed by Western blotting. Detection of FLAG‐tagged RIG‐I and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels. E. For analysis of NS1‐TRIM25 interaction HEK293 cells were infected with PR8/NS1‐T49, PR8/NS1‐T49A or PR8/NS1‐T49E (MOI of 1, 5 or 10 respectively) or were mock infected (control).Eighteen h p.i. cells were lysed and lysates subjected to immunoprecipitation with mouse anti‐TRIM25 antibody. TRIM25 and the co‐precipitated NS1 were detected by Western blotting using mouse anti‐TRIM25 antibody or mouse anti‐NS1 antibody. Detection of TRIM25 and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels.
    Figure Legend Snippet: Phosphorylation of NS1 T49 leads to reduced vRNA, RIG‐I and TRIM25 binding as well as structural destabilization of the RBD. A. A549 cells were transfected with pcDNA3 plasmids containing wt NS1 or NS1 with indicated mutations or were mock transfected. Cell lysates were subjected to immunoprecipitations 24 h p.i. using mouse anti‐NS1 antibody. Washed beads were incubated with vRNA and RNA bound to immunocomplexes was extracted. The relative amount of viral NS1 mRNA was determined by qRT‐PCR as described previously (Habjan et al ., 2008 ). Data represents mean ± SD of two independently repeated experiments. B. Comparative binding scores of RNA to RBD T49E, T49A and T49 as calculated using random forest model from the RNA–protein interaction prediction (RPISeq) tool (Muppirala et al ., 2011 ). The RNA sequence was obtained from Protein Data Bank (PDB) ID 2ZKO (Cheng et al ., 2009 ). C. Structural simulation on 2ZKO structure employing DUET (Pires et al ., 2014b ) was predicted as destabilizing (−0.17 Kcal/mol) resulting in an unfavourable RNA binding ability. The figure shows the stereo‐chemical effect of E49 compared with T49. The RNA helix is shown in yellow, while the two monomers of the NS1 dimer are shown in green and blue. NS1 residues T49 and E49 are shown in red and purple respectively. Molecular graphic simulation was performed using Bioblender (Andrei et al ., 2012 ). D. For analysis of NS1‐RIG‐I interaction, HEK293 cells transiently expressing FLAG‐tagged RIG‐I and the indicated NS1 proteins were subjected to crosslinking with DSP after 48 h, followed by quenching with glycine. Control cells were mock transfected. FLAG‐tagged RIG‐I was immunoprecipitated with anti‐FLAG M2 antibody. Detection of FLAG‐tagged RIG‐I and co‐precipitated NS1 protein was performed by Western blotting. Detection of FLAG‐tagged RIG‐I and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels. E. For analysis of NS1‐TRIM25 interaction HEK293 cells were infected with PR8/NS1‐T49, PR8/NS1‐T49A or PR8/NS1‐T49E (MOI of 1, 5 or 10 respectively) or were mock infected (control).Eighteen h p.i. cells were lysed and lysates subjected to immunoprecipitation with mouse anti‐TRIM25 antibody. TRIM25 and the co‐precipitated NS1 were detected by Western blotting using mouse anti‐TRIM25 antibody or mouse anti‐NS1 antibody. Detection of TRIM25 and NS1 in the cell lysates before immunoprecipitation served as ‘input control’ ensuring comparable expression levels.

    Techniques Used: Binding Assay, Transfection, Incubation, Quantitative RT-PCR, Sequencing, RNA Binding Assay, Expressing, Immunoprecipitation, Western Blot, Infection

    13) Product Images from "Control of CBP co-activating activity by arginine methylation"

    Article Title: Control of CBP co-activating activity by arginine methylation

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf548

    Fig. 4. CBP is methylated by CARM1 on R742 in vivo . ( A ) Left panel: the anti-methylated CBP antibody was tested for its specificity by a slot blot experiment with the indicated amount of various peptides. Right panel: 10 ng of GST–CBP FL and of GST–CBP 3R→A were methylated by GST–CARM1 (lanes 3 and 2, respectively) or not (lane 1). Reaction products were resolved onto an 8% SDS–PAGE followed by a western blot using the purified anti-methylated CBP antibody. ( B ) Immunoprecipitations from HeLa nuclear extracts (100 µl) were performed using an anti-HA polyclonal antibody (Irr.), an anti-CBP antibody and the affinity purified anti-methylated CBP antibody. Immunoprecipitates and the input (1 µl of HeLa nuclear extracts) were analysed by western blot using the anti-methylated CBP antibody. ( C ) Lysates of HeLa cells transfected either with control siRNA duplexes (CTR) or with specific CARM1 siRNA duplexes (CARM1) were tested by western blot for the expression level of CARM1, of HDAC-1, -2 and -3 (anti-HDAC3; Transduction Laboratories), of CBP and of CBP specifically methylated on R742.
    Figure Legend Snippet: Fig. 4. CBP is methylated by CARM1 on R742 in vivo . ( A ) Left panel: the anti-methylated CBP antibody was tested for its specificity by a slot blot experiment with the indicated amount of various peptides. Right panel: 10 ng of GST–CBP FL and of GST–CBP 3R→A were methylated by GST–CARM1 (lanes 3 and 2, respectively) or not (lane 1). Reaction products were resolved onto an 8% SDS–PAGE followed by a western blot using the purified anti-methylated CBP antibody. ( B ) Immunoprecipitations from HeLa nuclear extracts (100 µl) were performed using an anti-HA polyclonal antibody (Irr.), an anti-CBP antibody and the affinity purified anti-methylated CBP antibody. Immunoprecipitates and the input (1 µl of HeLa nuclear extracts) were analysed by western blot using the anti-methylated CBP antibody. ( C ) Lysates of HeLa cells transfected either with control siRNA duplexes (CTR) or with specific CARM1 siRNA duplexes (CARM1) were tested by western blot for the expression level of CARM1, of HDAC-1, -2 and -3 (anti-HDAC3; Transduction Laboratories), of CBP and of CBP specifically methylated on R742.

    Techniques Used: Methylation, In Vivo, Dot Blot, SDS Page, Western Blot, Purification, Affinity Purification, Transfection, Expressing, Transduction

    14) Product Images from "MCM-BP regulates unloading of the MCM2-7 helicase in late S phase"

    Article Title: MCM-BP regulates unloading of the MCM2-7 helicase in late S phase

    Journal: Genes & Development

    doi: 10.1101/gad.614411

    Xenopus MCM-BP binds to MCM proteins. ( A ) Western blot analysis of 0.5 μL of Xenopus interphase egg extracts with the rabbit polyclonal anti-MCM-BP antibody (I) or preimmune serum (PI). ( B ) MCM-BP associates with MCM proteins in Xenopus interphase egg extracts. Immunoprecipitations carried out using anti-MCM-BP (lane 3 ) or anti-MCM7 (lane 4 ) antibodies were analyzed by Western blotting using the indicated antibodies. Also shown is a mock immunoprecipitation to determine background signals (lane 2 ) and the proteins present in untreated egg extracts (0.5 μL) (lane 1 ). ( C ) MCM-BP and MCM3 immunoprecipitates from Xenopus interphase egg extracts were analyzed by MS. MS profiles were identified using the Mascot search engine. Score: Mascot scores.
    Figure Legend Snippet: Xenopus MCM-BP binds to MCM proteins. ( A ) Western blot analysis of 0.5 μL of Xenopus interphase egg extracts with the rabbit polyclonal anti-MCM-BP antibody (I) or preimmune serum (PI). ( B ) MCM-BP associates with MCM proteins in Xenopus interphase egg extracts. Immunoprecipitations carried out using anti-MCM-BP (lane 3 ) or anti-MCM7 (lane 4 ) antibodies were analyzed by Western blotting using the indicated antibodies. Also shown is a mock immunoprecipitation to determine background signals (lane 2 ) and the proteins present in untreated egg extracts (0.5 μL) (lane 1 ). ( C ) MCM-BP and MCM3 immunoprecipitates from Xenopus interphase egg extracts were analyzed by MS. MS profiles were identified using the Mascot search engine. Score: Mascot scores.

    Techniques Used: Western Blot, Immunoprecipitation, Mass Spectrometry

    15) Product Images from "GSK3 is a regulator of RAR-mediated differentiation"

    Article Title: GSK3 is a regulator of RAR-mediated differentiation

    Journal: Leukemia

    doi: 10.1038/leu.2012.2

    GSK3β binds RARα. ( a ) GSK3β can bind RARα. Co-immunoprecipitations were performed as indicated to test for the interaction of GSK3â and RARá in Hela cells treated for 6 h with AT (1 μ m ) or SB (30
    Figure Legend Snippet: GSK3β binds RARα. ( a ) GSK3β can bind RARα. Co-immunoprecipitations were performed as indicated to test for the interaction of GSK3â and RARá in Hela cells treated for 6 h with AT (1 μ m ) or SB (30

    Techniques Used:

    16) Product Images from "UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument"

    Article Title: UL88 Mediates the Incorporation of a Subset of Proteins into the Virion Tegument

    Journal: Journal of Virology

    doi: 10.1128/JVI.00474-20

    The composition of the tegument layer is altered in virions produced in the absence of UL88. (A) Western blot analysis of WT TB40/E, UL88-STOP, and UL88-REV purified on a sodium tartrate gradient. The blots were probed for the envelope protein gB and the tegument proteins UL88, UL47, UL48, pp71, pp150, UL71, pp28, and pp65. Gradient purifications were done for three distinct preparations of each virus, and the blots shown represent one of these preparations. (B) Western blot analysis of fibroblasts infected (MOI, 3) with WT TB40/E, UL88-STOP, UL88- galK , and UL88-REV at 96 hpi. The blots were probed with antibodies against the tegument proteins UL88, UL47, UL48, pp150, pp28, and pp65 and tubulin as a loading control. Results of quantitative analysis of the blots in panels A and B plus two additional repeats are shown to the right of the blots. Error bars represent standard deviations. (C) Quantitative PCR analysis of UL48 transcript levels at 96 hpi in fibroblasts infected with WT TB40/E, UL88-STOP, and UL88-REV. Values are relative to the wild-type sample after normalization and are averages for three biological replicates. (D) Western blot analysis of immunoprecipitations using FLAG beads of HEK293TN cell lysates prepared 24 h after cotransfection with UL88 and either UL47-FLAG or vector control. Blots were probed for FLAG and UL88. (E) Fibroblasts expressing a vector control or UL47-FLAG were infected with WT TB40/E (MOI, 3), and lysates were subjected to immunoprecipitation analysis with FLAG beads at 96 hpi. Blots were probed for antibodies against FLAG, UL88, and tubulin. *, IgG heavy chain; ‡, nonspecific band.
    Figure Legend Snippet: The composition of the tegument layer is altered in virions produced in the absence of UL88. (A) Western blot analysis of WT TB40/E, UL88-STOP, and UL88-REV purified on a sodium tartrate gradient. The blots were probed for the envelope protein gB and the tegument proteins UL88, UL47, UL48, pp71, pp150, UL71, pp28, and pp65. Gradient purifications were done for three distinct preparations of each virus, and the blots shown represent one of these preparations. (B) Western blot analysis of fibroblasts infected (MOI, 3) with WT TB40/E, UL88-STOP, UL88- galK , and UL88-REV at 96 hpi. The blots were probed with antibodies against the tegument proteins UL88, UL47, UL48, pp150, pp28, and pp65 and tubulin as a loading control. Results of quantitative analysis of the blots in panels A and B plus two additional repeats are shown to the right of the blots. Error bars represent standard deviations. (C) Quantitative PCR analysis of UL48 transcript levels at 96 hpi in fibroblasts infected with WT TB40/E, UL88-STOP, and UL88-REV. Values are relative to the wild-type sample after normalization and are averages for three biological replicates. (D) Western blot analysis of immunoprecipitations using FLAG beads of HEK293TN cell lysates prepared 24 h after cotransfection with UL88 and either UL47-FLAG or vector control. Blots were probed for FLAG and UL88. (E) Fibroblasts expressing a vector control or UL47-FLAG were infected with WT TB40/E (MOI, 3), and lysates were subjected to immunoprecipitation analysis with FLAG beads at 96 hpi. Blots were probed for antibodies against FLAG, UL88, and tubulin. *, IgG heavy chain; ‡, nonspecific band.

    Techniques Used: Produced, Western Blot, Purification, Infection, Real-time Polymerase Chain Reaction, Cotransfection, Plasmid Preparation, Expressing, Immunoprecipitation

    17) Product Images from "Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression"

    Article Title: Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003575

    Igo1 interacts with PP2A Cdc55 upon Ser64 phosphorylation in lateS/G2 phase of the cell cycle. A . Primary sequence alignment of endosulfines from different species around the serine phosphorylated by Greatwall/Rim15. B . Cell extracts from wild type and rim15 Δ cells expressing HA3-Cdc55 and Igo1-Pk3 as only sources of Cdc55 and Igo1 were subjected to immunoprecipitation with anti-Pk antibodies. The amount of HA3-Cdc55, Pph21, Tap42 and Rts3 co-immunoprecipitating with Igo1-Pk3 was assessed by western blot analysis. An extract from an Igo1-untagged strain was used as negative control (no tag). The asterisk marks the IgG in the IPs. C . Co-immunoprecipitations of HA3-Cdc55 with either wild type Igo1-myc8 or Igo1-S64A-myc8, in the presence or absence of RIM15 . D–E . Cycling (cyc) cultures of cells expressing HA3-Cdc55 and Igo1-Pk3 was arrested in G1 by α-factor and released in fresh medium at 25°C. At different time points after release cell samples were collected to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies (left, IP:αPk), the protein levels of HA3-Cdc55, Igo1-Pk3, Clb2, Cdc5 (left, Input), as well as the kinetics of budding, DNA replication (by FACS, data not shown), spindle formation/elongation and nuclear division (right graph). Pgk1 was used as loading control for the western blot. F . Cycling (cyc) cells were arrested in G1 by alpha factor or in S phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc) to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies. G . A cycling (cyc) culture of cells expressing Igo1-myc8 was arrested in G1 by alpha factor and released into the cell cycle at 25°C. At the indicated time points cell samples were collected for FACS analysis of DNA contents (not shown) and to prepare TCA protein extracts that were run on precast Phos-tag gels to visualize the phosphorylation of Igo1 by mobility shift. Extracts from rim15Δ and Igo1-S64A-myc8 cells were loaded as controls. Note that alpha factor induces an additional mobility shift likely corresponding to additional phosphorylations that are quickly lost upon cell cycle entry.
    Figure Legend Snippet: Igo1 interacts with PP2A Cdc55 upon Ser64 phosphorylation in lateS/G2 phase of the cell cycle. A . Primary sequence alignment of endosulfines from different species around the serine phosphorylated by Greatwall/Rim15. B . Cell extracts from wild type and rim15 Δ cells expressing HA3-Cdc55 and Igo1-Pk3 as only sources of Cdc55 and Igo1 were subjected to immunoprecipitation with anti-Pk antibodies. The amount of HA3-Cdc55, Pph21, Tap42 and Rts3 co-immunoprecipitating with Igo1-Pk3 was assessed by western blot analysis. An extract from an Igo1-untagged strain was used as negative control (no tag). The asterisk marks the IgG in the IPs. C . Co-immunoprecipitations of HA3-Cdc55 with either wild type Igo1-myc8 or Igo1-S64A-myc8, in the presence or absence of RIM15 . D–E . Cycling (cyc) cultures of cells expressing HA3-Cdc55 and Igo1-Pk3 was arrested in G1 by α-factor and released in fresh medium at 25°C. At different time points after release cell samples were collected to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies (left, IP:αPk), the protein levels of HA3-Cdc55, Igo1-Pk3, Clb2, Cdc5 (left, Input), as well as the kinetics of budding, DNA replication (by FACS, data not shown), spindle formation/elongation and nuclear division (right graph). Pgk1 was used as loading control for the western blot. F . Cycling (cyc) cells were arrested in G1 by alpha factor or in S phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc) to analyse the interaction between Igo1-Pk3 and HA3-Cdc55 after immunoprecipitation with anti-Pk antibodies. G . A cycling (cyc) culture of cells expressing Igo1-myc8 was arrested in G1 by alpha factor and released into the cell cycle at 25°C. At the indicated time points cell samples were collected for FACS analysis of DNA contents (not shown) and to prepare TCA protein extracts that were run on precast Phos-tag gels to visualize the phosphorylation of Igo1 by mobility shift. Extracts from rim15Δ and Igo1-S64A-myc8 cells were loaded as controls. Note that alpha factor induces an additional mobility shift likely corresponding to additional phosphorylations that are quickly lost upon cell cycle entry.

    Techniques Used: Sequencing, Expressing, Immunoprecipitation, Western Blot, Negative Control, FACS, Mobility Shift

    18) Product Images from "Squid, Cup and PABP 55B function together to regulate gurken translation in Drosophila"

    Article Title: Squid, Cup and PABP 55B function together to regulate gurken translation in Drosophila

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2007.11.008

    pAbp55B interacts with Enc biochemically and genetically (A) Immunoprecipitations were performed out of ovarian lysates in the presence or absence of RNase using α-Enc or α-Dorsal. The lysate lane represents 5% of the sample probed for interactions. Western blots were probed with α-PABP55B. (B) Heterozygosity for pAbp k10109 enhances the weakly ventralized phenotype of enc Q4 / enc Q4 and of enc UU3 / enc UU3 homozygotes at 25°C, increases the percentage of collapsed eggs, and decreases the overall number of eggs laid. Eggs were characterized as either wild-type like, weakly ventralized (appendages fused at the base), moderately ventralized (single, slender fused appendage), strongly ventralized (no appendage material), or collapsed. Binomial probabilities for the frequency of each eggshell morphology classification were calculated for enc Q4 / enc Q4 ; pAbp k10109 / + relative to enc Q4 / enc Q4 mutants and enc UU3 / enc UU3 ; pAbp k10109 / + relative to enc UU3 / enc UU3 mutants. †, p > 0.05; *, p
    Figure Legend Snippet: pAbp55B interacts with Enc biochemically and genetically (A) Immunoprecipitations were performed out of ovarian lysates in the presence or absence of RNase using α-Enc or α-Dorsal. The lysate lane represents 5% of the sample probed for interactions. Western blots were probed with α-PABP55B. (B) Heterozygosity for pAbp k10109 enhances the weakly ventralized phenotype of enc Q4 / enc Q4 and of enc UU3 / enc UU3 homozygotes at 25°C, increases the percentage of collapsed eggs, and decreases the overall number of eggs laid. Eggs were characterized as either wild-type like, weakly ventralized (appendages fused at the base), moderately ventralized (single, slender fused appendage), strongly ventralized (no appendage material), or collapsed. Binomial probabilities for the frequency of each eggshell morphology classification were calculated for enc Q4 / enc Q4 ; pAbp k10109 / + relative to enc Q4 / enc Q4 mutants and enc UU3 / enc UU3 ; pAbp k10109 / + relative to enc UU3 / enc UU3 mutants. †, p > 0.05; *, p

    Techniques Used: Western Blot

    Cup and PABP55B interact with Sqd in ovarian extracts (A) Immunoprecipitations were performed out of ovarian lysates using either α-Sqd or a negative control antibody, α-Dorsal. Specific Sqd interactors (marked by arrows) were excised from the gel and sequenced by mass spectrometry. Positively identified bands are labeled. (B-E) Immunoprecipitations were performed in the presence or absence of RNase (if indicated) using α-Sqd, α-Cup, α-PABP55B, or α-Dorsal. The lysate lane represents 5% of the sample probed for interactions. Western blots were probed with α-PABP55B (B and E) or α-Cup (C-E). These experiments demonstrate a strong interaction between Sqd, Cup, and PABP55B. (E) Additional immunoprecipitations were performed including α-Hrb27C/Hrp48 and probed with α-Cup and α-PABP55B. In addition to the regular exposure of the Cup western, an extended exposure is shown to clearly demonstrate the somewhat weaker interaction of Hrb27C/Hrp48 with Cup.
    Figure Legend Snippet: Cup and PABP55B interact with Sqd in ovarian extracts (A) Immunoprecipitations were performed out of ovarian lysates using either α-Sqd or a negative control antibody, α-Dorsal. Specific Sqd interactors (marked by arrows) were excised from the gel and sequenced by mass spectrometry. Positively identified bands are labeled. (B-E) Immunoprecipitations were performed in the presence or absence of RNase (if indicated) using α-Sqd, α-Cup, α-PABP55B, or α-Dorsal. The lysate lane represents 5% of the sample probed for interactions. Western blots were probed with α-PABP55B (B and E) or α-Cup (C-E). These experiments demonstrate a strong interaction between Sqd, Cup, and PABP55B. (E) Additional immunoprecipitations were performed including α-Hrb27C/Hrp48 and probed with α-Cup and α-PABP55B. In addition to the regular exposure of the Cup western, an extended exposure is shown to clearly demonstrate the somewhat weaker interaction of Hrb27C/Hrp48 with Cup.

    Techniques Used: Negative Control, Mass Spectrometry, Labeling, Western Blot

    19) Product Images from "CK2 Protein Kinase Is Stimulated and Redistributed by Functional Herpes Simplex Virus ICP27 Protein"

    Article Title: CK2 Protein Kinase Is Stimulated and Redistributed by Functional Herpes Simplex Virus ICP27 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.7.4315-4325.2003

    HSV-1 infection activates CK2 by 3 h postinfection. (A) Extracts of cells at different times postinfection with wt HSV-1 (KOS strain) were applied to a polyacrylamide gel containing dephosphorylated casein and subjected to electrophoresis. The gel was denatured, renatured, and then incubated with kinase reaction buffer in the presence of [γ- 32 P]ATP and, after extensive washes, was dried and autoradiographed. (B) Western blot analysis, using the polyclonal Rα403 antibody against CK2 α and α′ subunits (upper panel) and the polyclonal β-c antibody against the β subunit (lower panel), of the CK2 subunits at different times postinfection with wt HSV-1. (C) Using extracts from mock- or wt-infected cells and the polyclonal antibody β-c against the CK2 β subunit, immunoprecipitations were performed at 4 h postinfection. Western blot analysis was performed using the Rα405 polyclonal antibody against the CK2 α and α′ subunits. The intense ∼50-kDa band corresponds to the heavy chain of the antibody used for immunoprecipitation.
    Figure Legend Snippet: HSV-1 infection activates CK2 by 3 h postinfection. (A) Extracts of cells at different times postinfection with wt HSV-1 (KOS strain) were applied to a polyacrylamide gel containing dephosphorylated casein and subjected to electrophoresis. The gel was denatured, renatured, and then incubated with kinase reaction buffer in the presence of [γ- 32 P]ATP and, after extensive washes, was dried and autoradiographed. (B) Western blot analysis, using the polyclonal Rα403 antibody against CK2 α and α′ subunits (upper panel) and the polyclonal β-c antibody against the β subunit (lower panel), of the CK2 subunits at different times postinfection with wt HSV-1. (C) Using extracts from mock- or wt-infected cells and the polyclonal antibody β-c against the CK2 β subunit, immunoprecipitations were performed at 4 h postinfection. Western blot analysis was performed using the Rα405 polyclonal antibody against the CK2 α and α′ subunits. The intense ∼50-kDa band corresponds to the heavy chain of the antibody used for immunoprecipitation.

    Techniques Used: Infection, Electrophoresis, Incubation, Western Blot, Immunoprecipitation

    20) Product Images from "The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts with the Dopamine Transporter"

    Article Title: The Multiple LIM Domain-Containing Adaptor Protein Hic-5 Synaptically Colocalizes and Interacts with the Dopamine Transporter

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.22-16-07045.2002

    The full-length DAT interacts with Hic-5 in HEK293 cells. HEK293 cells were transfected with the HA-tagged human DAT and the myc-tagged mouse Hic-5 individually or in combination. A , Immunoprecipitations ( IP ) with the anti-HA antibody and Western blot detection ( IB ) with a polyclonal anti-Hic-5 antibody. Hic-5-myc is immunoprecipitated with the anti-HA antibody only when coexpressed with DAT-HA. B , Immunoprecipitations using the anti-myc antibody and Western blot detection with the rat anti-DAT antibody. DAT-HA is immunoprecipitated with the anti-myc antibody only when it is coexpressed with Hic-5-myc.
    Figure Legend Snippet: The full-length DAT interacts with Hic-5 in HEK293 cells. HEK293 cells were transfected with the HA-tagged human DAT and the myc-tagged mouse Hic-5 individually or in combination. A , Immunoprecipitations ( IP ) with the anti-HA antibody and Western blot detection ( IB ) with a polyclonal anti-Hic-5 antibody. Hic-5-myc is immunoprecipitated with the anti-HA antibody only when coexpressed with DAT-HA. B , Immunoprecipitations using the anti-myc antibody and Western blot detection with the rat anti-DAT antibody. DAT-HA is immunoprecipitated with the anti-myc antibody only when it is coexpressed with Hic-5-myc.

    Techniques Used: Hydrophobic Interaction Chromatography, Transfection, Western Blot, Immunoprecipitation

    21) Product Images from "Lysine Acetyltransferase GCN5b Interacts with AP2 Factors and Is Required for Toxoplasma gondii Proliferation"

    Article Title: Lysine Acetyltransferase GCN5b Interacts with AP2 Factors and Is Required for Toxoplasma gondii Proliferation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003830

    Reciprocal immunoprecipitation confirms the in vivo interaction of GCN5b with endogenously HA-tagged AP2IX-7 and AP2X-8. A. IFAs showing localization of each AP2 to the parasite nucleus. Anti-HA (green) shows localization of designate HA-tagged AP2 protein; DAPI (blue) co-stains the nuclei. B. Immunoprecipitations using an anti-HA antibody were performed on parasite lysates made from AP2IX-7 HA , AP2X-8 HA , and AP2X-5 HA parasites, as well as the parental RHΔ ku80 line. The immunoprecipitated complexes were analyzed by Western blot using antibodies recognizing anti-HA, GCN5b, or β-tubulin. Arrowheads designate the expected size of each tagged AP2 protein (these AP2 proteins are very large and various breakdown products were observed in the different conditions used to process lysates versus IPs).
    Figure Legend Snippet: Reciprocal immunoprecipitation confirms the in vivo interaction of GCN5b with endogenously HA-tagged AP2IX-7 and AP2X-8. A. IFAs showing localization of each AP2 to the parasite nucleus. Anti-HA (green) shows localization of designate HA-tagged AP2 protein; DAPI (blue) co-stains the nuclei. B. Immunoprecipitations using an anti-HA antibody were performed on parasite lysates made from AP2IX-7 HA , AP2X-8 HA , and AP2X-5 HA parasites, as well as the parental RHΔ ku80 line. The immunoprecipitated complexes were analyzed by Western blot using antibodies recognizing anti-HA, GCN5b, or β-tubulin. Arrowheads designate the expected size of each tagged AP2 protein (these AP2 proteins are very large and various breakdown products were observed in the different conditions used to process lysates versus IPs).

    Techniques Used: Immunoprecipitation, In Vivo, Western Blot

    22) Product Images from "Dynamics of the peroxisomal import cycle of PpPex20p"

    Article Title: Dynamics of the peroxisomal import cycle of PpPex20p

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200508096

    In vivo interaction of Pex20p with known peroxins and Fox3p. Immunoprecipitations (IP) were performed with the HA antibody (α-HA) on the indicated lysates and were immunoblotted (IB) with the indicated antibody. (A) Lysates of Δpex7 and Δpex14 cells expressing HA-Pex7p or of wild-type (WT; PPY12) cells. (B) Lysates of Δpex20 or Δpex7 cells expressing HA-Pex20p. (C) Lysates from Δpex20 cells or from HA-Pex20p–expressing Δpex20 and Δpex14 cells.
    Figure Legend Snippet: In vivo interaction of Pex20p with known peroxins and Fox3p. Immunoprecipitations (IP) were performed with the HA antibody (α-HA) on the indicated lysates and were immunoblotted (IB) with the indicated antibody. (A) Lysates of Δpex7 and Δpex14 cells expressing HA-Pex7p or of wild-type (WT; PPY12) cells. (B) Lysates of Δpex20 or Δpex7 cells expressing HA-Pex20p. (C) Lysates from Δpex20 cells or from HA-Pex20p–expressing Δpex20 and Δpex14 cells.

    Techniques Used: In Vivo, Expressing

    23) Product Images from "Inhibition of PLK1-dependent EBNA2 phosphorylation promotes lymphomagenesis in EBV-infected mice"

    Article Title: Inhibition of PLK1-dependent EBNA2 phosphorylation promotes lymphomagenesis in EBV-infected mice

    Journal: bioRxiv

    doi: 10.1101/2021.03.29.437455

    EBNA2/PLK1 phosphorylation-dependent complex formation Transfection and immunoprecipitation of (A) HA-tagged or for smaller fragments (B) GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB). (C) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site (UniProt ID: P12978.1). The panel on the right summarizes the results of the co-immunoprecipitations in (A) and (B). (D) Multiple sequence alignment (left) and superposition (right) of several phosphopeptides present in published crystal structures of PLK1 PBD (phosphorylated residues stained red). Crystal structures of the PBD in complex with peptides show that the positively charged groove of PBD docks in a similar mode to the negatively charged phosphopeptides. References for PDB ID: 1Q4K ( Cheng et al , 2003 ), 1UMW ( Elia et al. , 2003a ), 4E9C ( Śledź et al , 2012 ), 3C5L ( Yun et al , 2009 ), 3HIK ( Yun et al. , 2009 ), 5X3S ( Lee et al , 2018 ), 3Q1I ( Pavlovsky et al , 2012 ). Potential residues of EBNA2, which might be a PBD docking site, are listed below. (E) Immunoprecipitation of HA-tagged EBNA2 mutant ST266AV, TSS377VAA and SPSS467APAA using HA-specific antibodies. (F) ITC thermogram of PLK1 PBD titrated with the peptide PNTSSPS or the phosphopeptide PNTSpSPS of EBNA2. (G) GST-pulldown of PLK1 from total cellular extracts using Cyclin B/CDK1 phosphorylated GST-EBNA2 region 342-422. (H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.
    Figure Legend Snippet: EBNA2/PLK1 phosphorylation-dependent complex formation Transfection and immunoprecipitation of (A) HA-tagged or for smaller fragments (B) GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB). (C) Schematic outline of EBNA2, its dimerization domains (END, DIM), the region used by CBF1 to recruit EBNA2 to DNA (WW), the C-terminal transactivation domain (TAD), the nuclear localization signal (N) and the EBNA2 fragments used to map the PLK1 docking site (UniProt ID: P12978.1). The panel on the right summarizes the results of the co-immunoprecipitations in (A) and (B). (D) Multiple sequence alignment (left) and superposition (right) of several phosphopeptides present in published crystal structures of PLK1 PBD (phosphorylated residues stained red). Crystal structures of the PBD in complex with peptides show that the positively charged groove of PBD docks in a similar mode to the negatively charged phosphopeptides. References for PDB ID: 1Q4K ( Cheng et al , 2003 ), 1UMW ( Elia et al. , 2003a ), 4E9C ( Śledź et al , 2012 ), 3C5L ( Yun et al , 2009 ), 3HIK ( Yun et al. , 2009 ), 5X3S ( Lee et al , 2018 ), 3Q1I ( Pavlovsky et al , 2012 ). Potential residues of EBNA2, which might be a PBD docking site, are listed below. (E) Immunoprecipitation of HA-tagged EBNA2 mutant ST266AV, TSS377VAA and SPSS467APAA using HA-specific antibodies. (F) ITC thermogram of PLK1 PBD titrated with the peptide PNTSSPS or the phosphopeptide PNTSpSPS of EBNA2. (G) GST-pulldown of PLK1 from total cellular extracts using Cyclin B/CDK1 phosphorylated GST-EBNA2 region 342-422. (H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Sequencing, Staining, Mutagenesis

    PLK1 phosphorylates the EBNA2 residue S457 and T465 within the C-terminal transactivation domain (A) Doxycycline (Dox) induction of EBNA2 in DG75 Dox HA-EBNA2 cells treated for 24 hours. Co-immunoprecipitates of HA-EBNA2 and endogenous PLK1 are visualized by Western blotting. (B) EBNA2/PLK1 co-precipitates were submitted to kinase reactions using [γ- 32 P] ATP in the absence (control) or presence of 50 ng recombinant active PLK1. (C) EBNA2 co-precipitates were submitted to kinase reactions in the absence (control) or the presence of the PLK1 inhibitor Volasertib (40 nM). (D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting. (E) Immunoprecipitates were submitted to kinase reactions as in B, but samples were treated with Volasertib (40 nM) (+) or treated with solvent only (-). (F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ- 32 P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate ( Yuan et al , 2002 ), was used as a positive control. (G) Schematic presentation of EBNA2 phosphorylation sites by CDK1 and PLK1. (H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity. (I) GST-pull down assay using GST-EBNA2 446-474 as a bait to purify cellular proteins from DG75 cells followed by Western blotting and quantification of signals obtained by GST and p300 specific antibodies. Relative binding affinities (rel. p300 bindg.) are normalized to the wt signal.
    Figure Legend Snippet: PLK1 phosphorylates the EBNA2 residue S457 and T465 within the C-terminal transactivation domain (A) Doxycycline (Dox) induction of EBNA2 in DG75 Dox HA-EBNA2 cells treated for 24 hours. Co-immunoprecipitates of HA-EBNA2 and endogenous PLK1 are visualized by Western blotting. (B) EBNA2/PLK1 co-precipitates were submitted to kinase reactions using [γ- 32 P] ATP in the absence (control) or presence of 50 ng recombinant active PLK1. (C) EBNA2 co-precipitates were submitted to kinase reactions in the absence (control) or the presence of the PLK1 inhibitor Volasertib (40 nM). (D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting. (E) Immunoprecipitates were submitted to kinase reactions as in B, but samples were treated with Volasertib (40 nM) (+) or treated with solvent only (-). (F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ- 32 P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate ( Yuan et al , 2002 ), was used as a positive control. (G) Schematic presentation of EBNA2 phosphorylation sites by CDK1 and PLK1. (H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity. (I) GST-pull down assay using GST-EBNA2 446-474 as a bait to purify cellular proteins from DG75 cells followed by Western blotting and quantification of signals obtained by GST and p300 specific antibodies. Relative binding affinities (rel. p300 bindg.) are normalized to the wt signal.

    Techniques Used: Western Blot, Recombinant, Binding Assay, Mutagenesis, In Vitro, Positive Control, Activation Assay, Luciferase, Plasmid Preparation, Activity Assay, Pull Down Assay

    24) Product Images from "α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production"

    Article Title: α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-017-1704-z

    α-Synuclein is a MAM protein and binds to VAPB but not PTPIP51 in immunoprecipitation assays. a Immunoblots of total lysates (Lys), MAM, mitochondria (Mit) and ER proteins from rat brains. Samples were probed for α-synuclein plus FACL4, VAPB and calnexin as MAM/ER markers, and PTPIP51 and HSP60 as mitochondrial markers. Extended exposures of immunoblots reveal PTPIP51 signal in total lysate samples. b , c Immunoprecipitation assays of α-synuclein binding to PTPIP51 and VAPB. HEK293 cells were transfected with either PTPIP51-HA ( b ) or myc-VAPB ( c ) + either empty control vector (CTRL), α-synuclein, α-synucleinA53T or α-synucleinA30P as indicated. PTPIP51 was immunoprecipitated using rabbit anti-HA and detected on immunoblots with mouse anti-HA; α-synuclein was detected with mouse anti-α-synuclein. VAPB was immunoprecipitated using mouse anti-myc and detected on immunoblots with rabbit anti-HA; α-synuclein was detected with rabbit anti-α-synuclein. Both inputs and immunoprecipitations (IP) are shown. α-Synuclein displayed no binding to PTPIP51 but bound to VAPB. Bar chart in c shows relative levels of α-synuclein bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. α-Synuclein signals were normalized to immunoprecipitated VAPB-myc signals. Data are expressed as percentage of the wild-type α-synuclein signal and were analysed by one-way ANOVA and Tukey’s post hoc test. N = 4; error bars are SEM, * p > 0.05, ** p
    Figure Legend Snippet: α-Synuclein is a MAM protein and binds to VAPB but not PTPIP51 in immunoprecipitation assays. a Immunoblots of total lysates (Lys), MAM, mitochondria (Mit) and ER proteins from rat brains. Samples were probed for α-synuclein plus FACL4, VAPB and calnexin as MAM/ER markers, and PTPIP51 and HSP60 as mitochondrial markers. Extended exposures of immunoblots reveal PTPIP51 signal in total lysate samples. b , c Immunoprecipitation assays of α-synuclein binding to PTPIP51 and VAPB. HEK293 cells were transfected with either PTPIP51-HA ( b ) or myc-VAPB ( c ) + either empty control vector (CTRL), α-synuclein, α-synucleinA53T or α-synucleinA30P as indicated. PTPIP51 was immunoprecipitated using rabbit anti-HA and detected on immunoblots with mouse anti-HA; α-synuclein was detected with mouse anti-α-synuclein. VAPB was immunoprecipitated using mouse anti-myc and detected on immunoblots with rabbit anti-HA; α-synuclein was detected with rabbit anti-α-synuclein. Both inputs and immunoprecipitations (IP) are shown. α-Synuclein displayed no binding to PTPIP51 but bound to VAPB. Bar chart in c shows relative levels of α-synuclein bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. α-Synuclein signals were normalized to immunoprecipitated VAPB-myc signals. Data are expressed as percentage of the wild-type α-synuclein signal and were analysed by one-way ANOVA and Tukey’s post hoc test. N = 4; error bars are SEM, * p > 0.05, ** p

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

    25) Product Images from "Fidelity of G protein ?-subunit association by the G protein ?-subunit-like domains of RGS6, RGS7, and RGS11"

    Article Title: Fidelity of G protein ?-subunit association by the G protein ?-subunit-like domains of RGS6, RGS7, and RGS11

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

    doi:

    In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.
    Figure Legend Snippet: In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

    Techniques Used: In Vitro, Binding Assay, Sequencing, Mutagenesis, Immunoprecipitation, SDS Page, Autoradiography

    26) Product Images from "Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate"

    Article Title: Arginine Methylation by PRMT1 Regulates Muscle Stem Cell Fate

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00457-16

    Eya1 is a PRMT1 substrate. (A) Candidate peptides were arrayed for major myogenic determinant proteins, and the filter was incubated with recombinant GST-PRMT1 and [ 3 H] S -adenosylmethionine for in vitro methylation assay. The 3 H-positive spots represent the methylated peptides. Peptides from histones H2B and H4 were used as positive controls. A complete list of the peptides is found in Table 1 . (B) HEK293T cells were cotransfected with MYC epitope-tagged PRMT1 and FLAG-tagged Eya1 or FLAG-YFP as control. Cell lysates were prepared, and immunoprecipitations were performed with M2–anti-FLAG beads. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG, MYC, and ASYM26. TCL, total cell lysates; IB, immunoblot; IP, immunoprecipitation. (C) HEK293T cells were transfected with siLuciferase (siLuc) or siPRMT1 for 24 h. Then, cells were transfected with expression vectors for FLAG-Eya1 or FLAG-YFP. Cell lysates were prepared 48 h later, and coimmunoprecipitations were performed as described for panel B. See also Tables 1 and 2 .
    Figure Legend Snippet: Eya1 is a PRMT1 substrate. (A) Candidate peptides were arrayed for major myogenic determinant proteins, and the filter was incubated with recombinant GST-PRMT1 and [ 3 H] S -adenosylmethionine for in vitro methylation assay. The 3 H-positive spots represent the methylated peptides. Peptides from histones H2B and H4 were used as positive controls. A complete list of the peptides is found in Table 1 . (B) HEK293T cells were cotransfected with MYC epitope-tagged PRMT1 and FLAG-tagged Eya1 or FLAG-YFP as control. Cell lysates were prepared, and immunoprecipitations were performed with M2–anti-FLAG beads. The bound proteins were separated by SDS-PAGE and immunoblotted with anti-FLAG, MYC, and ASYM26. TCL, total cell lysates; IB, immunoblot; IP, immunoprecipitation. (C) HEK293T cells were transfected with siLuciferase (siLuc) or siPRMT1 for 24 h. Then, cells were transfected with expression vectors for FLAG-Eya1 or FLAG-YFP. Cell lysates were prepared 48 h later, and coimmunoprecipitations were performed as described for panel B. See also Tables 1 and 2 .

    Techniques Used: Incubation, Recombinant, In Vitro, Methylation, SDS Page, Immunoprecipitation, Transfection, Expressing

    27) Product Images from "The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans"

    Article Title: The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks748

    Slicing ALG-1 is required to generate functional miRISC. ( A ) Detection of lin-4 in bound (IP) or unbound (FT) fractions after ALG-1 immunoprecipitations. Transgenically expressed ALG-1 wild-type (wt) or catalytically defective (AAA) protein were IPed from alg-1(lf) ; alg-2(RNAi) animals. The presence of lin-4 strands was detected by Northern blotting (left panel) and 10% of immunopurified complexes were used for detection of ALG-1 (right panel). ( B ) The quantification of three independent experiments is shown in the lower panel. Errors bars represent a 95% confidence interval and a Student’s two-sided t -test was applied to obtain P values.
    Figure Legend Snippet: Slicing ALG-1 is required to generate functional miRISC. ( A ) Detection of lin-4 in bound (IP) or unbound (FT) fractions after ALG-1 immunoprecipitations. Transgenically expressed ALG-1 wild-type (wt) or catalytically defective (AAA) protein were IPed from alg-1(lf) ; alg-2(RNAi) animals. The presence of lin-4 strands was detected by Northern blotting (left panel) and 10% of immunopurified complexes were used for detection of ALG-1 (right panel). ( B ) The quantification of three independent experiments is shown in the lower panel. Errors bars represent a 95% confidence interval and a Student’s two-sided t -test was applied to obtain P values.

    Techniques Used: Functional Assay, Northern Blot

    28) Product Images from "NF-κB-induced KIAA1199 promotes survival through EGFR signalling"

    Article Title: NF-κB-induced KIAA1199 promotes survival through EGFR signalling

    Journal: Nature Communications

    doi: 10.1038/ncomms6232

    KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).
    Figure Legend Snippet: KIAA1199 connects Semaphorin 3A signalling to EGFR phosphorylation. ( a ) Semaphorin 3A-mediated EGFR phosphorylation requires KIAA1199. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with Semaphorin 3A (100 ng ml −1 ) and WB analyses were carried out on the resulting cell extracts (lysis in SDS 1%). ( b ) KIAA1199 deficiency does not have an impact on EGFR mRNA levels in cervical cancer cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR, to assess EGFR mRNA levels. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from two independent experiments (means±s.d.) are shown. ( c ) Plexin A2 deficiency potentiates Semaphorin 3A-mediated EGFR phosphorylation. Control or Plexin A2-depleted CaSki cells were left untreated or stimulated with Semaphorin 3A for the indicated periods of time. The resulting cell extracts (lysis in SDS 1%) were subjected to WBs using the indicated antibodies. ( d ) Plexin A2 deficiency prolongs the binding of KIAA1199 to EGFR on EGF stimulation. Control or Plexin A2-deficient cells were untreated or stimulated with EGF for the indicated periods of time. Cell extracts were subjected to anti-FLAG (negative control) or -EGFR immunoprecipitations followed by anti- KIAA1199 or -EGFR WBs (top panels). Crude cell extracts were subjected to anti-pEGFR (Y845 and Y1068) (to validate the triggering of the EGF-dependent pathway), -EGFR, -KIAA1199, -Plexin A2 and -HSP90 WBs, as indicated. ( e ) KIAA1199 binds EGFR through its N-terminal domain. Cells were transfected with the indicated expression plasmids and protein extracts were subjected to anti-HA (negative control) or -FLAG IPs followed by an anti-EGFR WB (top panel). Crude cell extracts were also subjected to anti-EGFR and -FLAG WB analyses (bottom panels).

    Techniques Used: Western Blot, Lysis, shRNA, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Transfection, Expressing

    29) Product Images from "UPF1 helicase promotes TSN-mediated miRNA decay"

    Article Title: UPF1 helicase promotes TSN-mediated miRNA decay

    Journal: Genes & Development

    doi: 10.1101/gad.303537.117

    Evidence that TSN and UPF1 form a complex in HEK293T cells. ( A ) Western blotting of formaldehyde cross-linked HEK293T cells that stably express both Flag-TSN and MYC-UPF1 or, as a negative control, Flag and MYC. Western blotting was performed before (−) or after immunoprecipitation using anti-Flag in the absence (−) or presence (+) of RNase I. Here and elsewhere, lanes below the wedge analyze threefold dilutions of lysate. ( B ) As in A except that immunoprecipitations were performed using anti-MYC. ( C ) As in A except that lysates of untransfected HEK293T cells were used, and immunoprecipitations were performed using anti-TSN or, as a negative control, rabbit IgG (rIgG). ( D ) As in C except that immunoprecipitations were performed using anti-UPF1 or, as a negative control, nonimmunized rabbit serum (NRS). ( E ) HIS pull-downs of Escherichia coli -produced HIS-TSN or HIS-EGFP (HIS-tagged enhanced green fluorescence protein) in the presence of baculovirus-produced Flag-UPF1. Results are representative of three or more independently performed experiments.
    Figure Legend Snippet: Evidence that TSN and UPF1 form a complex in HEK293T cells. ( A ) Western blotting of formaldehyde cross-linked HEK293T cells that stably express both Flag-TSN and MYC-UPF1 or, as a negative control, Flag and MYC. Western blotting was performed before (−) or after immunoprecipitation using anti-Flag in the absence (−) or presence (+) of RNase I. Here and elsewhere, lanes below the wedge analyze threefold dilutions of lysate. ( B ) As in A except that immunoprecipitations were performed using anti-MYC. ( C ) As in A except that lysates of untransfected HEK293T cells were used, and immunoprecipitations were performed using anti-TSN or, as a negative control, rabbit IgG (rIgG). ( D ) As in C except that immunoprecipitations were performed using anti-UPF1 or, as a negative control, nonimmunized rabbit serum (NRS). ( E ) HIS pull-downs of Escherichia coli -produced HIS-TSN or HIS-EGFP (HIS-tagged enhanced green fluorescence protein) in the presence of baculovirus-produced Flag-UPF1. Results are representative of three or more independently performed experiments.

    Techniques Used: Western Blot, Stable Transfection, Negative Control, Immunoprecipitation, Produced, Fluorescence

    30) Product Images from "α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production"

    Article Title: α-Synuclein binds to the ER–mitochondria tethering protein VAPB to disrupt Ca2+ homeostasis and mitochondrial ATP production

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-017-1704-z

    α-Synuclein is a MAM protein and binds to VAPB but not PTPIP51 in immunoprecipitation assays. a Immunoblots of total lysates (Lys), MAM, mitochondria (Mit) and ER proteins from rat brains. Samples were probed for α-synuclein plus FACL4, VAPB and calnexin as MAM/ER markers, and PTPIP51 and HSP60 as mitochondrial markers. Extended exposures of immunoblots reveal PTPIP51 signal in total lysate samples. b , c Immunoprecipitation assays of α-synuclein binding to PTPIP51 and VAPB. HEK293 cells were transfected with either PTPIP51-HA ( b ) or myc-VAPB ( c ) + either empty control vector (CTRL), α-synuclein, α-synucleinA53T or α-synucleinA30P as indicated. PTPIP51 was immunoprecipitated using rabbit anti-HA and detected on immunoblots with mouse anti-HA; α-synuclein was detected with mouse anti-α-synuclein. VAPB was immunoprecipitated using mouse anti-myc and detected on immunoblots with rabbit anti-HA; α-synuclein was detected with rabbit anti-α-synuclein. Both inputs and immunoprecipitations (IP) are shown. α-Synuclein displayed no binding to PTPIP51 but bound to VAPB. Bar chart in c shows relative levels of α-synuclein bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. α-Synuclein signals were normalized to immunoprecipitated VAPB-myc signals. Data are expressed as percentage of the wild-type α-synuclein signal and were analysed by one-way ANOVA and Tukey’s post hoc test. N = 4; error bars are SEM, * p > 0.05, ** p
    Figure Legend Snippet: α-Synuclein is a MAM protein and binds to VAPB but not PTPIP51 in immunoprecipitation assays. a Immunoblots of total lysates (Lys), MAM, mitochondria (Mit) and ER proteins from rat brains. Samples were probed for α-synuclein plus FACL4, VAPB and calnexin as MAM/ER markers, and PTPIP51 and HSP60 as mitochondrial markers. Extended exposures of immunoblots reveal PTPIP51 signal in total lysate samples. b , c Immunoprecipitation assays of α-synuclein binding to PTPIP51 and VAPB. HEK293 cells were transfected with either PTPIP51-HA ( b ) or myc-VAPB ( c ) + either empty control vector (CTRL), α-synuclein, α-synucleinA53T or α-synucleinA30P as indicated. PTPIP51 was immunoprecipitated using rabbit anti-HA and detected on immunoblots with mouse anti-HA; α-synuclein was detected with mouse anti-α-synuclein. VAPB was immunoprecipitated using mouse anti-myc and detected on immunoblots with rabbit anti-HA; α-synuclein was detected with rabbit anti-α-synuclein. Both inputs and immunoprecipitations (IP) are shown. α-Synuclein displayed no binding to PTPIP51 but bound to VAPB. Bar chart in c shows relative levels of α-synuclein bound to VAPB in the immunoprecipitations following quantification of signals from immunoblots. α-Synuclein signals were normalized to immunoprecipitated VAPB-myc signals. Data are expressed as percentage of the wild-type α-synuclein signal and were analysed by one-way ANOVA and Tukey’s post hoc test. N = 4; error bars are SEM, * p > 0.05, ** p

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

    31) Product Images from "Control of myogenesis by rodent SINE-containing lncRNAs"

    Article Title: Control of myogenesis by rodent SINE-containing lncRNAs

    Journal: Genes & Development

    doi: 10.1101/gad.212639.112

    Confirmation that the predicted m½-sbsRNA–mRNA duplexes function as SBSs in C2C12 MBs. ( A ) Diagrams of pFLUC reporter plasmids, the 3′ UTR of which contains no SBS or the denoted mRNA 3′ UTR SINE situated 224 nt downstream from the FLUC termination codon. The cross-hatched box represents the FLUC open translational reading frame. ( B ) Western blotting using lysates of C2C12 MBs prior to immunoprecipitation. MBs (4 × 10 6 per 150-mm dish) had been transiently transfected with 10 μg of p STAU1-HA 3 or pUC19, 1 μg of each of the seven FLUC reporter plasmids, and 2 μg of phCMV-MUP and formaldehyde-cross-linked prior to lysis. ( C ) Western blotting ( top ) or RT–PCR ( bottom ) of lysates analyzed in B before (−) or after immunoprecipitation (IP) using anti-HA (α-HA) or, as a control for nonspecific immunoprecipitation, mIgG. ( D ) Diagrams of plasmids encoding m½-sbsRNA1(B1), m½-sbsRNA1(B1) harboring 12 copies of the MS2bs, or FLUC mRNA harboring 12 copies of the MS2bs. ( E ) Western blotting ( top ) or RT–PCR ( bottom ) before (−) or after immunoprecipitation of lysates of formaldehyde-cross-linked C2C12 MBs (4 × 10 6 per 150-mm dish) that had been transiently transfected with 50 nM specified siRNA and, 1 d later, with 5 μg of pFlag-MS2-hMGFP, 1 μg of each of the seven reporter plasmids shown in A, 2 μg of p hCMV-MUP , and the denoted plasmid encoding m½-sbsRNA1(B1), m½-sbsRNA1(B1)-MS2bs, or FLUC-MS2bs. Immunoprecipitations were performed using anti-Flag or mIgG. All results are representative of at least two independently performed experiments (Supplemental Fig. 4; Supplemental Table 4).
    Figure Legend Snippet: Confirmation that the predicted m½-sbsRNA–mRNA duplexes function as SBSs in C2C12 MBs. ( A ) Diagrams of pFLUC reporter plasmids, the 3′ UTR of which contains no SBS or the denoted mRNA 3′ UTR SINE situated 224 nt downstream from the FLUC termination codon. The cross-hatched box represents the FLUC open translational reading frame. ( B ) Western blotting using lysates of C2C12 MBs prior to immunoprecipitation. MBs (4 × 10 6 per 150-mm dish) had been transiently transfected with 10 μg of p STAU1-HA 3 or pUC19, 1 μg of each of the seven FLUC reporter plasmids, and 2 μg of phCMV-MUP and formaldehyde-cross-linked prior to lysis. ( C ) Western blotting ( top ) or RT–PCR ( bottom ) of lysates analyzed in B before (−) or after immunoprecipitation (IP) using anti-HA (α-HA) or, as a control for nonspecific immunoprecipitation, mIgG. ( D ) Diagrams of plasmids encoding m½-sbsRNA1(B1), m½-sbsRNA1(B1) harboring 12 copies of the MS2bs, or FLUC mRNA harboring 12 copies of the MS2bs. ( E ) Western blotting ( top ) or RT–PCR ( bottom ) before (−) or after immunoprecipitation of lysates of formaldehyde-cross-linked C2C12 MBs (4 × 10 6 per 150-mm dish) that had been transiently transfected with 50 nM specified siRNA and, 1 d later, with 5 μg of pFlag-MS2-hMGFP, 1 μg of each of the seven reporter plasmids shown in A, 2 μg of p hCMV-MUP , and the denoted plasmid encoding m½-sbsRNA1(B1), m½-sbsRNA1(B1)-MS2bs, or FLUC-MS2bs. Immunoprecipitations were performed using anti-Flag or mIgG. All results are representative of at least two independently performed experiments (Supplemental Fig. 4; Supplemental Table 4).

    Techniques Used: Western Blot, Immunoprecipitation, Transfection, Lysis, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

    32) Product Images from "Balance between Acetylation and Methylation of Histone H3 Lysine 9 on the E2F-Responsive Dihydrofolate Reductase Promoter"

    Article Title: Balance between Acetylation and Methylation of Histone H3 Lysine 9 on the E2F-Responsive Dihydrofolate Reductase Promoter

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.5.1614-1622.2003

    Specific variations in histone H3 modification on the DHFR promoter during G 1 phase progression. (A) Cell cycle analysis of NIH 3T3 cells starved for serum for 48 h and then reinduced with serum for the indicated time. (B) Chromatin from NIH 3T3 cells in G 0 or G 1 /S was immunoprecipitated using antibodies specific for histone H3 acetylated on K9 (K9 Ac), histone H3 acetylated on K14 (K14 Ac), or histone H3 methylated on K9 (K9 Met). The amount of the DHFR and GAPDH promoters present in immunoprecipitations from G 0 (d0 or g0, respectively) or G 1 /S cells (d1 or g1, respectively) was calculated relative to the input (as described in Materials and Methods). The relative enrichment was obtained by the following formula: relative enrichment = (d x /d0)/(g x /g0) ( x is 0 or 1). The means of two (K9 Ac and K14 Ac) or three (K9 Met) entirely independent experiments are shown.
    Figure Legend Snippet: Specific variations in histone H3 modification on the DHFR promoter during G 1 phase progression. (A) Cell cycle analysis of NIH 3T3 cells starved for serum for 48 h and then reinduced with serum for the indicated time. (B) Chromatin from NIH 3T3 cells in G 0 or G 1 /S was immunoprecipitated using antibodies specific for histone H3 acetylated on K9 (K9 Ac), histone H3 acetylated on K14 (K14 Ac), or histone H3 methylated on K9 (K9 Met). The amount of the DHFR and GAPDH promoters present in immunoprecipitations from G 0 (d0 or g0, respectively) or G 1 /S cells (d1 or g1, respectively) was calculated relative to the input (as described in Materials and Methods). The relative enrichment was obtained by the following formula: relative enrichment = (d x /d0)/(g x /g0) ( x is 0 or 1). The means of two (K9 Ac and K14 Ac) or three (K9 Met) entirely independent experiments are shown.

    Techniques Used: Modification, Cell Cycle Assay, Immunoprecipitation, Methylation

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    Transfection:

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    Lysis:

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    Protease Inhibitor:

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    Polymerase Chain Reaction:

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    Centrifugation:

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    Incubation:

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    Western Blot:

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    SDS Page:

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    Concentration Assay:

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    Roche immunoprecipitation cells
    EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) <t>Co-immunoprecipitation</t> (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p
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    Roche immunoprecipitations
    Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line <t>immunoprecipitations</t> by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and
    Immunoprecipitations, 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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    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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    EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p

    Journal: PLoS ONE

    Article Title: MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition

    doi: 10.1371/journal.pone.0170798

    Figure Lengend Snippet: EGFR-MET interaction is modulated by EGFR mutations. (A) Western blot (WB) of total MET levels in H1975 derived cells. Tubulin levels are shown as loading control. Values beneath blots are relative levels of MET compared to the total levels in the H1975 L858R/T790M cell line from 2 independent experiments +/−SD (B) MET (7q31) (red signal) copy number analysis by FISH in the three H1975 cell lines using the Leica Kreatech C-MET (7q31)/SE7 FISH probe (KBI-10719). The green signal indicates the chromosome 7 centromere control probe. Scale bar 10 mm. Average copy number and ratio between MET and chromosome 7 centromere probe are also indicated (n = 30 cells). (C) Immunofluorescence of total EGFR (Alexa546 –red in the image) and MET (Cyanine 5 –green in the image) in H1975 derived cells. Hoescht dye was used to stain the nuclei of the cells. Merge panels are also shown. Bars, 20 μm. (D) Co-immunoprecipitation (IP) of EGFR in H1975 derived cell lines. The EGFR antibody was used to immunoprecipitate. EGFR and MET levels are shown in both bound and input fractions. The gels shown in the figure were run separately for the bound and input fractions, as indicated by the dotted line, under the same experimental conditions. (E) Fluorescence lifetime imaging was performed on cells plated to sub-confluence on cover-slips and time-resolved analysis in Tri2. Quantification of average FRET efficiency (*** p

    Article Snippet: Immunoprecipitation Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 10 mM NaF, 1 mM Na3 VO4 , 10 mM N-ethylmaleimide, 0.01 μM Calyculin A) with Protease inhibitor cocktail set I (Roche).

    Techniques: Western Blot, Derivative Assay, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Immunoprecipitation, Fluorescence, Imaging

    Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line immunoprecipitations by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and

    Journal: Autophagy

    Article Title: Characterization of early autophagy signaling by quantitative phosphoproteomics

    doi: 10.4161/auto.26864

    Figure Lengend Snippet: Figure 6. Large-scale screen for phosphorylated LC3 interaction partners. ( A ) Using a rat eGFP-MAP1LC3-MCF7 cell line immunoprecipitations by anti-GFP antibodies were performed to enrich LC3-interacting proteins from untreated control cells and

    Article Snippet: For autophagosome accumulation cells were treated with 2 nM conA (Sigma-Aldrich, 27689) for 7 h. Anti-GFP (Santa Cruz Biotechnology, sc-9996) immunoprecipitations of SILAC-labeled MCF7 cells expressing rat eGFP-MAP1LC3 were performed in modified RIPA buffer containing 1% NP-40, 150 mM NaCl, 0.25% Na deoxycholate, 50 mM Tris pH 7.5 and Complete Protease Inhibitor tablets (Roche, 11836145001).

    Techniques:

    In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

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

    Article Title: Fidelity of G protein ?-subunit association by the G protein ?-subunit-like domains of RGS6, RGS7, and RGS11

    doi:

    Figure Lengend Snippet: In vitro G β binding specificity of GGL domain mutants. ( A ) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G γ 2 ) are plotted above the primary sequence of the GGL domain ( x axis). α-Helices within G γ 2 ) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 ( B ), RGS7 ( C ), and RGS11 proteins ( D–F ). HA-tagged RGS or G γ proteins were immunoprecipitated in low detergent (except as noted in E ) with anti-HA mAb, and associated G β proteins were visualized by SDS/PAGE and autoradiography. ( E ) Immunoprecipitations (IP) of cotranslated G β 5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

    Article Snippet: “Low-detergent” immunoprecipitations were performed and washed in buffer D [50 mM NaCl/10 mM MgCl2 /50 mM Tris, pH 8.0/1 mM EDTA/10 mM 2-mercaptoethanol/20% (vol/vol) glycerol/Complete protease inhibitors; Roche Diagnostics] containing 0.05% C12E10, whereas “high-detergent” immunoprecipitations were performed in buffer D containing 0.1% Triton X-100 and washed in RIPA-500 buffer containing 500 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS.

    Techniques: In Vitro, Binding Assay, Sequencing, Mutagenesis, Immunoprecipitation, SDS Page, Autoradiography

    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