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GE Healthcare antigen antibody complexes
Antigen Antibody Complexes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 573 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 573 article reviews
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Western Blot:

Article Title: P4HA3 is Epigenetically Activated by Slug in Gastric Cancer and its Deregulation is Associated With Enhanced Metastasis and Poor Survival
Article Snippet: .. Antigen–antibody complexes were detected by electrochemiluminiscence Western blotting detection reagent (GE Healthcare, Marlborough, Massachusetts). .. Band densitometry was performed using ImageJ software (version 1.48, Bio-Rad, Hercules, California, USA).

Incubation:

Article Title: TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development
Article Snippet: .. Antigen—antibody complexes were precipitated with protein A Sepharose (GE Healthcare) after overnight incubation at 4°C. ..

other:

Article Title: Fat‐specific protein 27α inhibits autophagy‐dependent lipid droplet breakdown in white adipocytes
Article Snippet: Immune complexes were detected with horseradish peroxidase‐conjugated secondary antibodies and enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA).

Article Title: Epstein-Barr virus BARF1-induced NFκB/miR-146a/SMAD4 alterations in stomach cancer cells
Article Snippet: The antigen-antibody complexes were visualized using ECL-staining (Amersham, Arlington Heights, IL, USA) and expose to X-ray film.

Article Title: Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway
Article Snippet: Antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Article Title: Human Immunodeficiency Virus Induces a Dual Regulation of Bcl-2, Resulting in Persistent Infection of CD4+ T- or Monocytic Cell Lines
Article Snippet: The antigen-antibody complexes were revealed by enhanced chemiluminescence (ECL; Amersham).

Article Title: Targeting prohibitins at the cell surface prevents Th17‐mediated autoimmunity
Article Snippet: Antigen–antibody complexes were detected by horseradish peroxidase‐coupled secondary antibodies followed by enhanced chemiluminescence (Amersham Biosciences, Millipore, Thermo Scientific).

Article Title: The antineoplastic drug, trastuzumab, dysregulates metabolism in iPSC-derived cardiomyocytes
Article Snippet: After three washes with TBST, antigen–antibody complexes were detected with the ECL Plus chemiluminescent system (Amersham Biosciences) and visualized with film.

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  • 85
    GE Healthcare anti hnrnp r sera
    <t>hnRNP</t> R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.
    Anti Hnrnp R Sera, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hnrnp r sera/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    90
    GE Healthcare co immunoprecipitation assays eif3 complexes
    The octameric right leg subunits eIF3k and eIF3l impact their mutual expression and are dispensable for the integrity of the rest of <t>eIF3,</t> and the octameric right arm subunit eIF3e stabilizes binding of the right leg subunits (k, l) and eIF3d to the octamer, as well as the octamer attachment to the YLC. Protein levels of all eIF3 subunits and other eIFs upon knock down of either eIF3k, l or e were determined by Western blotting ( A, C, E , respectively). GAPDH was used as a loading control. To assess the integrity of eIF3, the anti-eIF3b <t>co-immunoprecipitation</t> experiments were carried out for each knock-down and the immunoprecipitated eIF3 subunits along with eIF3b were detected by Western blotting ( B, D, F ). The octameric eIF3 subunits are arranged at the left-hand side of each panel and non-octameric subunits are at the right-hand side. The YLC subunits are grouped and highlighted in bold. Quantifications of at least five independent experiments with standard deviations are shown in Supplementary Figure S4 and Table 1 .
    Co Immunoprecipitation Assays Eif3 Complexes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/co immunoprecipitation assays eif3 complexes/product/GE Healthcare
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    85
    GE Healthcare gst fused bcl gl protein
    MELK phosphorylates <t>Bcl-G</t> L in vitro . (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-G L , and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase <t>(GST)</t> Bcl-G L fusion recombinant protein (GST-Bcl-G L ) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-G L protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-G L (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-G L (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-G L proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.
    Gst Fused Bcl Gl Protein, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare α5 integrin immuno complexes
    Model for the roles of talin and kindlin during inside-out and outside-in signaling of α5β1 <t>integrin.</t> Integrin subunits are modelled according to Zhu et al. (2008) , with the <t>α5</t> subunit in green and the β1 subunit in blue showing the bent and clasped low affinity and the extended and unclasped high affinity conformations; the 9EG7 epitope is marked as red dot at the β1 leg and the FN ligand as beige dimers. ( A ) α5β1 integrin fails to shift from a bent to an extended/unclasped, high affinity state in the absence of talin-1/2 or kindlin-1/2; the bent/clasped conformation brings the EGF-2 domain of the β subunit in close contact with the calf domain of the α5 subunit and prevents exposure of the 9EG7 epitope. ( B ) In the absence of talin (Tln Ko ) and presence of Mn 2+ , kindlin-2 allows adhesion by stabilizing the high affinity conformation of a low number of integrins and the direct binding of paxillin, leading to nucleation of integrins, recruitment of FAK, FAK-dependent signaling and lamellipodia formation. ( C ) In the absence of kindlins (Kind Ko ), talin stabilizes the high affinity conformation of a low number of integrins but does not enable paxillin recruitment and lamellipodia formation. ( D ) In normal fibroblasts, binding of kindlin and talin to the β1 tail is associated with the stabilisation of the unclasped α5β1 heterodimer and 9EG7 epitope exposure. ( E ) Kindlin recruits paxillin and FAK through the kindlin-PH domain and ILK/Pinch/Parvin (IPP; not shown) in a talin-independent manner and induces cell spreading, proliferation and survival. ( F ) The high affinity conformation of α5β1 integrin is stabilized by linkage of the β1 tail to the actin cytoskeleton through talin (and potentially the IPP complex; not shown). The arrow length indicates integrin conformations existing at equilibrium. EGF, epidermal growth factor; FAK, focal adhesion kinase; FN, fibronectin; ILK, integrin-linked kinase; IPP, integrin-linked kinase-Pinch-Parvin; SFK, src family kinases. DOI: http://dx.doi.org/10.7554/eLife.10130.028
    α5 Integrin Immuno Complexes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: Labeling, Hybridization, Autoradiography, Rnase Protection Assay, Produced, Incubation

    hnRNP R is required for transcriptional induction of the c-fos gene. (A) Mouse C3H10T1/2 cells were treated by siRNA for hnRNP R (hnRNP R) or control siRNA (control), and the levels of hnRNP R were analyzed by Western blotting using an anti-hnRNP R antibody. The cells that were not treated with siRNA are indicated by (-). (B) Total cellular RNAs were prepared from the siRNA-treated cells, and RT-qPCR assays were used to determine the effect of hnRNP R knockdown on the induction of the c-fos gene after serum stimulation. The expression levels of the α-tubulin gene were used to normalize those of the c-fos gene at each time point, and the expression levels before serum stimulation were used to calculate fold activation at each time point. The values were averaged from five independent experiments, and the error bars represent S.E. The followings are the p values when comparing the c-fos expression level after hnRNP R knockdown versus that after control knockdown at each time point: p = 0.028 (10 min), p = 0.006 (20 min), p = 0.007 (30 min), p = 0.012 (45 min), p = 0.049 (60 min), p = 0.05 (90 min) and p = 0.01 (12 0min). (C) The ratios of the c-fos expression level after hnRNP R knockdown against that after control knockdown were calculated. The followings are the p values when comparing the ratio at each time point versus that at 10min: p = 0.404 (20 min), p = 0.107 (30 min), p = 0.001 (45 min), p = 0.002 (60 min), p = 0.0006 (90 min) and p = 0.0002 (120 min). Two asterisks (**) indicate p

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: hnRNP R is required for transcriptional induction of the c-fos gene. (A) Mouse C3H10T1/2 cells were treated by siRNA for hnRNP R (hnRNP R) or control siRNA (control), and the levels of hnRNP R were analyzed by Western blotting using an anti-hnRNP R antibody. The cells that were not treated with siRNA are indicated by (-). (B) Total cellular RNAs were prepared from the siRNA-treated cells, and RT-qPCR assays were used to determine the effect of hnRNP R knockdown on the induction of the c-fos gene after serum stimulation. The expression levels of the α-tubulin gene were used to normalize those of the c-fos gene at each time point, and the expression levels before serum stimulation were used to calculate fold activation at each time point. The values were averaged from five independent experiments, and the error bars represent S.E. The followings are the p values when comparing the c-fos expression level after hnRNP R knockdown versus that after control knockdown at each time point: p = 0.028 (10 min), p = 0.006 (20 min), p = 0.007 (30 min), p = 0.012 (45 min), p = 0.049 (60 min), p = 0.05 (90 min) and p = 0.01 (12 0min). (C) The ratios of the c-fos expression level after hnRNP R knockdown against that after control knockdown were calculated. The followings are the p values when comparing the ratio at each time point versus that at 10min: p = 0.404 (20 min), p = 0.107 (30 min), p = 0.001 (45 min), p = 0.002 (60 min), p = 0.0006 (90 min) and p = 0.0002 (120 min). Two asterisks (**) indicate p

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: Western Blot, Quantitative RT-PCR, Expressing, Activation Assay

    The efficiency of transcription reinitiation depends on the length of the transcripts. (A) In vitro transcription was performed using the pfMC2AT templates harboring a G-free cassette that ranges from 350 to 50 nt in length. The numbers above each lane indicate the length of the G-free cassette. Mediator and hnRNP R were added as indicated in the figure. A representative data of three experiments is shown. (B) Quantification of the first-round transcripts and the second-round transcripts in the presence of Mediator alone (“Mediator ONLY”) or both Mediator and hnRNP R (“Mediator(+), hnRNP R(+)”). To obtain the relative molar ratios of the transcripts, the measured transcript levels were normalized by the number of C residues within the transcribed region because [α- 32 P] CTP was used for labeling the transcripts. The molar ratios of the second-round transcripts to the first-round transcripts were calculated using the normalized transcript levels. All the values were averaged from three independent experiments, and error bars represent S.E. The number below each lane indicates the length of the G-free cassette. The p values of the molar ratio (2 nd /1 st ) on each template versus that on the pfMC2AT(350) are as follows. In the presence of only Mediator: p = 0.83 (pfMC2AT300), p = 0.21 (pfMC2AT250), p = 0.16 (pfMC2AT200), p = 0.13 (pfMC2AT150) and p = 0.13 (pfMC2AT100). In the presence of both Mediator and hnRNP R: p = 0.34 (pfMC2AT300), p = 0.042 (pfMC2AT250), p = 0.018 (pfMC2AT200), p = 0.008 (pfMC2AT150) and p = 0.022 (pfMC2AT100).

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: The efficiency of transcription reinitiation depends on the length of the transcripts. (A) In vitro transcription was performed using the pfMC2AT templates harboring a G-free cassette that ranges from 350 to 50 nt in length. The numbers above each lane indicate the length of the G-free cassette. Mediator and hnRNP R were added as indicated in the figure. A representative data of three experiments is shown. (B) Quantification of the first-round transcripts and the second-round transcripts in the presence of Mediator alone (“Mediator ONLY”) or both Mediator and hnRNP R (“Mediator(+), hnRNP R(+)”). To obtain the relative molar ratios of the transcripts, the measured transcript levels were normalized by the number of C residues within the transcribed region because [α- 32 P] CTP was used for labeling the transcripts. The molar ratios of the second-round transcripts to the first-round transcripts were calculated using the normalized transcript levels. All the values were averaged from three independent experiments, and error bars represent S.E. The number below each lane indicates the length of the G-free cassette. The p values of the molar ratio (2 nd /1 st ) on each template versus that on the pfMC2AT(350) are as follows. In the presence of only Mediator: p = 0.83 (pfMC2AT300), p = 0.21 (pfMC2AT250), p = 0.16 (pfMC2AT200), p = 0.13 (pfMC2AT150) and p = 0.13 (pfMC2AT100). In the presence of both Mediator and hnRNP R: p = 0.34 (pfMC2AT300), p = 0.042 (pfMC2AT250), p = 0.018 (pfMC2AT200), p = 0.008 (pfMC2AT150) and p = 0.022 (pfMC2AT100).

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: In Vitro, Labeling

    hnRNP R interacts with Mediator and other PIC components, as well as with the transcript RNA. GST pull-down assays were performed to test interaction between hnRNP R and Mediator or PC4 (A), interactions between hnRNP R and the activators (B) and interactions between hnRNP R and GTFs or Pol II (C). (D) The 32 P-labeled transcript RNA derived from the 390-nt G-free cassette was used to test interaction between hnRNP R and the transcript RNA. GST alone (A–D) and FLAG-tagged luciferase (A) were used as negative controls. (E) Immunoprecipitation was performed using HeLa nuclear extract with the anti-hnRNP R antibody. The proteins precipitated together with hnRNP R were analyzed by western blotting (WB) using anti-Elk-1 and anti-CREB antibodies. (F) RNA was extracted from the immunoprecipitate from HeLa nuclear extract with or without the anti-hnRNP R antibody, and then amplified by RT-PCR and analyzed on a 1% agarose gel.

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: hnRNP R interacts with Mediator and other PIC components, as well as with the transcript RNA. GST pull-down assays were performed to test interaction between hnRNP R and Mediator or PC4 (A), interactions between hnRNP R and the activators (B) and interactions between hnRNP R and GTFs or Pol II (C). (D) The 32 P-labeled transcript RNA derived from the 390-nt G-free cassette was used to test interaction between hnRNP R and the transcript RNA. GST alone (A–D) and FLAG-tagged luciferase (A) were used as negative controls. (E) Immunoprecipitation was performed using HeLa nuclear extract with the anti-hnRNP R antibody. The proteins precipitated together with hnRNP R were analyzed by western blotting (WB) using anti-Elk-1 and anti-CREB antibodies. (F) RNA was extracted from the immunoprecipitate from HeLa nuclear extract with or without the anti-hnRNP R antibody, and then amplified by RT-PCR and analyzed on a 1% agarose gel.

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: Labeling, Derivative Assay, Luciferase, Immunoprecipitation, Western Blot, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    hnRNP R and Mediator enhance transcription from the c- fos promoter in a cooperative manner. (A) In vitro transcription was performed in a reaction containing TFIIA (10 ng), TFIIB (10 ng), FLAG-tagged TFIID (F:TFIID) (which contained ∼0.1 ng of TBP), TFIIE (10 ng), TFIIF (20 ng), TFIIH (∼20 ng), Pol II (∼100 ng), F:SRF (20 ng), F:Elk-1 (10 ng), F:CREB (20 ng), F:ATF1 (20 ng) and PC4 (100 ng) in the presence (+) or absence (–) of hnRNP R (100 ng) and 3xF:Mediator (∼20 ng) as shown in the figure. pfMC2AT (100 ng), which harbors the c- fos promoter and the 390-nt G-free cassette, was used as a template. The reaction was performed in the presence of 0.2 mM ATP, 0.2 mM UTP, 12.5 µM CTP, 0.1 mM 3′-O-methyl GTP and 10 µCi of [α- 32 P] CTP. The arrow indicates the position of the 390-nt transcript (lanes 1–4), and the arrowhead indicates the shorter transcript observed in the presence of both Mediator and hnRNP R (lane 4). SRE, FAP-1, CRE and TATA indicate the serum response element, c-fos AP-1 site, cAMP-responsive element and TATA box, respectively. (B) In vitro transcription was performed as described in (A), except that the reaction contained the template, pfMC2AT(150), which harbors the 150-nt G-free cassette instead of the 390-nt G-free cassette. The arrow and arrowhead indicate the positions of the 150-nt transcript and the shorter transcript, respectively.

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: hnRNP R and Mediator enhance transcription from the c- fos promoter in a cooperative manner. (A) In vitro transcription was performed in a reaction containing TFIIA (10 ng), TFIIB (10 ng), FLAG-tagged TFIID (F:TFIID) (which contained ∼0.1 ng of TBP), TFIIE (10 ng), TFIIF (20 ng), TFIIH (∼20 ng), Pol II (∼100 ng), F:SRF (20 ng), F:Elk-1 (10 ng), F:CREB (20 ng), F:ATF1 (20 ng) and PC4 (100 ng) in the presence (+) or absence (–) of hnRNP R (100 ng) and 3xF:Mediator (∼20 ng) as shown in the figure. pfMC2AT (100 ng), which harbors the c- fos promoter and the 390-nt G-free cassette, was used as a template. The reaction was performed in the presence of 0.2 mM ATP, 0.2 mM UTP, 12.5 µM CTP, 0.1 mM 3′-O-methyl GTP and 10 µCi of [α- 32 P] CTP. The arrow indicates the position of the 390-nt transcript (lanes 1–4), and the arrowhead indicates the shorter transcript observed in the presence of both Mediator and hnRNP R (lane 4). SRE, FAP-1, CRE and TATA indicate the serum response element, c-fos AP-1 site, cAMP-responsive element and TATA box, respectively. (B) In vitro transcription was performed as described in (A), except that the reaction contained the template, pfMC2AT(150), which harbors the 150-nt G-free cassette instead of the 390-nt G-free cassette. The arrow and arrowhead indicate the positions of the 150-nt transcript and the shorter transcript, respectively.

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: In Vitro

    Functional domains of hnRNP R required for facilitating initiation and reinitiation. (A) Schematic diagram of the domain structure of hnRNP R and its deletion mutants are shown. hnRNP R is comprised of the acidic domain (acidic), three RNA recognition motifs (RRMs), a nuclear localization signal (NLS), arginine-glycine-glycine-rich domain (RGG) and the glutamine-asparagine-rich domain (QN). The results of RNA binding assays (shown in B) and in vitro transcription (shown in C) for the mutants are summarized on the right. An in vitro transcription assay for Δ1-446 was not done (nd) because the mutant was insoluble unless expressed as a GST-fusion protein. The domains required for initiation (1st round) and reinitiation (multiple round) are indicated above the diagram. (B) Binding of the hnRNP R to the RNA derived from the 390-nt G-free cassette was assayed using immobilized GST (lane 3), GST-hnRNP R (lane 4), or GST-hnRNP R mutants (lanes 5–10). The bound RNA was analyzed by electrophoresis and autoradiography, and lanes 1 and 2 contained 10% and 5% of the input RNA. (C) In vitro transcription was preformed using the purified FLAG-tagged hnRNP R mutants. pfMC2AT harboring the 390-nt G-free cassette was used as a template. The positions of the first-round and second-round transcripts are indicated by arrows on the right, and the relative levels of the first-round transcript are indicated below each lane.

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: Functional domains of hnRNP R required for facilitating initiation and reinitiation. (A) Schematic diagram of the domain structure of hnRNP R and its deletion mutants are shown. hnRNP R is comprised of the acidic domain (acidic), three RNA recognition motifs (RRMs), a nuclear localization signal (NLS), arginine-glycine-glycine-rich domain (RGG) and the glutamine-asparagine-rich domain (QN). The results of RNA binding assays (shown in B) and in vitro transcription (shown in C) for the mutants are summarized on the right. An in vitro transcription assay for Δ1-446 was not done (nd) because the mutant was insoluble unless expressed as a GST-fusion protein. The domains required for initiation (1st round) and reinitiation (multiple round) are indicated above the diagram. (B) Binding of the hnRNP R to the RNA derived from the 390-nt G-free cassette was assayed using immobilized GST (lane 3), GST-hnRNP R (lane 4), or GST-hnRNP R mutants (lanes 5–10). The bound RNA was analyzed by electrophoresis and autoradiography, and lanes 1 and 2 contained 10% and 5% of the input RNA. (C) In vitro transcription was preformed using the purified FLAG-tagged hnRNP R mutants. pfMC2AT harboring the 390-nt G-free cassette was used as a template. The positions of the first-round and second-round transcripts are indicated by arrows on the right, and the relative levels of the first-round transcript are indicated below each lane.

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: Functional Assay, RNA Binding Assay, In Vitro, Mutagenesis, Binding Assay, Derivative Assay, Electrophoresis, Autoradiography, Purification

    The Mediator containing the CDK8 module does not facilitate transcription reinitiation. (A) Med(0.5) and Med(0.85) were purified from HeLa nuclear extract expressing 3xFLAG-tagged MED10 by using phosphocellulose (P11) and anti-FLAG® M2 affinity gel. The purified Med(0.5) and Med(0.85) were separated by SDS-PAGE and silver-stained. The positions of the molecular mass marker are indicated on the left. Mediator was detected via 3xFLAG-tagged MED10 using an anti-FLAG antibody. (B) Western blot analyses were performed using anti-CDK8, anti-Cyclin C, anti-MED12, anti-MED13 or anti-MED17 antibody. (C)  In vitro  transcription was performed as described in   Figure 1A . hnRNP R, Med(0.5) and Med(0.85) were added as shown in the figure. The arrows indicate the first-round (1st) and second-round (2nd) transcripts, and the values (means ± SE) of the quantified data from four independent experiments are shown in the top (1st round) and middle (2nd round) panels on the right. The bottom panel on the right shows the ratios of the second-round to first-round transcripts.

    Journal: PLoS ONE

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    doi: 10.1371/journal.pone.0072496

    Figure Lengend Snippet: The Mediator containing the CDK8 module does not facilitate transcription reinitiation. (A) Med(0.5) and Med(0.85) were purified from HeLa nuclear extract expressing 3xFLAG-tagged MED10 by using phosphocellulose (P11) and anti-FLAG® M2 affinity gel. The purified Med(0.5) and Med(0.85) were separated by SDS-PAGE and silver-stained. The positions of the molecular mass marker are indicated on the left. Mediator was detected via 3xFLAG-tagged MED10 using an anti-FLAG antibody. (B) Western blot analyses were performed using anti-CDK8, anti-Cyclin C, anti-MED12, anti-MED13 or anti-MED17 antibody. (C) In vitro transcription was performed as described in Figure 1A . hnRNP R, Med(0.5) and Med(0.85) were added as shown in the figure. The arrows indicate the first-round (1st) and second-round (2nd) transcripts, and the values (means ± SE) of the quantified data from four independent experiments are shown in the top (1st round) and middle (2nd round) panels on the right. The bottom panel on the right shows the ratios of the second-round to first-round transcripts.

    Article Snippet: The purified recombinant human hnRNP R was used to immunize rabbits, and the obtained anti-hnRNP R sera were affinity purified by an antigen column, in which Glutathione S-transferase (GST)-hnRNP R was cross-linked to Glutathione Sepharose 4B (GE healthcare).

    Techniques: Purification, Expressing, SDS Page, Staining, Marker, Western Blot, In Vitro

    The octameric right leg subunits eIF3k and eIF3l impact their mutual expression and are dispensable for the integrity of the rest of eIF3, and the octameric right arm subunit eIF3e stabilizes binding of the right leg subunits (k, l) and eIF3d to the octamer, as well as the octamer attachment to the YLC. Protein levels of all eIF3 subunits and other eIFs upon knock down of either eIF3k, l or e were determined by Western blotting ( A, C, E , respectively). GAPDH was used as a loading control. To assess the integrity of eIF3, the anti-eIF3b co-immunoprecipitation experiments were carried out for each knock-down and the immunoprecipitated eIF3 subunits along with eIF3b were detected by Western blotting ( B, D, F ). The octameric eIF3 subunits are arranged at the left-hand side of each panel and non-octameric subunits are at the right-hand side. The YLC subunits are grouped and highlighted in bold. Quantifications of at least five independent experiments with standard deviations are shown in Supplementary Figure S4 and Table 1 .

    Journal: Nucleic Acids Research

    Article Title: Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer

    doi: 10.1093/nar/gkw972

    Figure Lengend Snippet: The octameric right leg subunits eIF3k and eIF3l impact their mutual expression and are dispensable for the integrity of the rest of eIF3, and the octameric right arm subunit eIF3e stabilizes binding of the right leg subunits (k, l) and eIF3d to the octamer, as well as the octamer attachment to the YLC. Protein levels of all eIF3 subunits and other eIFs upon knock down of either eIF3k, l or e were determined by Western blotting ( A, C, E , respectively). GAPDH was used as a loading control. To assess the integrity of eIF3, the anti-eIF3b co-immunoprecipitation experiments were carried out for each knock-down and the immunoprecipitated eIF3 subunits along with eIF3b were detected by Western blotting ( B, D, F ). The octameric eIF3 subunits are arranged at the left-hand side of each panel and non-octameric subunits are at the right-hand side. The YLC subunits are grouped and highlighted in bold. Quantifications of at least five independent experiments with standard deviations are shown in Supplementary Figure S4 and Table 1 .

    Article Snippet: Co-Immunoprecipitation assays eIF3 complexes were immunoprecipitated from WCEs, the preparation of which is described above, using GammaBind G Sepharose (GE Healthcare, cat # 17-0885-01) with either anti-eIF3b (Santa Cruz, cat # sc-16377) or anti-eIF3f (kind gift of Dr Hiroaki Imataka) primary antibodies.

    Techniques: Expressing, Binding Assay, Western Blot, Immunoprecipitation

    MELK phosphorylates Bcl-G L in vitro . (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-G L , and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-G L fusion recombinant protein (GST-Bcl-G L ) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-G L protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-G L (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-G L (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-G L proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.

    Journal: Breast Cancer Research

    Article Title: Involvement of maternal embryonic leucine zipper kinase (MELK) in mammary carcinogenesis through interaction with Bcl-G, a pro-apoptotic member of the Bcl-2 family

    doi: 10.1186/bcr1650

    Figure Lengend Snippet: MELK phosphorylates Bcl-G L in vitro . (a) Immunoprecipitates were subjected to immune complex kinase assay with wild-type (WT)-MELK or kinase-dead (D150A)-MELK). The single arrowhead indicates phosphorylated Bcl-G L , and the double arrowhead points to an autophosphorylated MELK protein. (b) Phosphorylation of a bacterial glutathione S-transferase (GST) Bcl-G L fusion recombinant protein (GST-Bcl-G L ) by His-tagged WT-MELK (WT). The single arrowhead indicates phosphorylated GST-Bcl-G L protein, and the double arrowhead indicates an autophosphorylated His-tagged MELK protein. (c) In vitro phosphorylation of various partial amino-terminal constructs of Bcl-G L (N-1, N-2, N-3 and N-4; see Figure 3e) and full-length Bcl-G L (FL) by MELK. The single arrowheads indicate phosphorylated immunoprecipitated Bcl-G L proteins, and the double arrowhead indicates an autophosphorylated MELK recombinant protein.

    Article Snippet: For confirmation of direct binding of BCL-GL and MELK, we removed GST from GST-fused BCL-GL protein using PreScission protease (GE Healthcare) according to the supplier's instructions.

    Techniques: In Vitro, Immune Complex Kinase Assay, Recombinant, Construct, Immunoprecipitation

    Model for the roles of talin and kindlin during inside-out and outside-in signaling of α5β1 integrin. Integrin subunits are modelled according to Zhu et al. (2008) , with the α5 subunit in green and the β1 subunit in blue showing the bent and clasped low affinity and the extended and unclasped high affinity conformations; the 9EG7 epitope is marked as red dot at the β1 leg and the FN ligand as beige dimers. ( A ) α5β1 integrin fails to shift from a bent to an extended/unclasped, high affinity state in the absence of talin-1/2 or kindlin-1/2; the bent/clasped conformation brings the EGF-2 domain of the β subunit in close contact with the calf domain of the α5 subunit and prevents exposure of the 9EG7 epitope. ( B ) In the absence of talin (Tln Ko ) and presence of Mn 2+ , kindlin-2 allows adhesion by stabilizing the high affinity conformation of a low number of integrins and the direct binding of paxillin, leading to nucleation of integrins, recruitment of FAK, FAK-dependent signaling and lamellipodia formation. ( C ) In the absence of kindlins (Kind Ko ), talin stabilizes the high affinity conformation of a low number of integrins but does not enable paxillin recruitment and lamellipodia formation. ( D ) In normal fibroblasts, binding of kindlin and talin to the β1 tail is associated with the stabilisation of the unclasped α5β1 heterodimer and 9EG7 epitope exposure. ( E ) Kindlin recruits paxillin and FAK through the kindlin-PH domain and ILK/Pinch/Parvin (IPP; not shown) in a talin-independent manner and induces cell spreading, proliferation and survival. ( F ) The high affinity conformation of α5β1 integrin is stabilized by linkage of the β1 tail to the actin cytoskeleton through talin (and potentially the IPP complex; not shown). The arrow length indicates integrin conformations existing at equilibrium. EGF, epidermal growth factor; FAK, focal adhesion kinase; FN, fibronectin; ILK, integrin-linked kinase; IPP, integrin-linked kinase-Pinch-Parvin; SFK, src family kinases. DOI: http://dx.doi.org/10.7554/eLife.10130.028

    Journal: eLife

    Article Title: Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

    doi: 10.7554/eLife.10130

    Figure Lengend Snippet: Model for the roles of talin and kindlin during inside-out and outside-in signaling of α5β1 integrin. Integrin subunits are modelled according to Zhu et al. (2008) , with the α5 subunit in green and the β1 subunit in blue showing the bent and clasped low affinity and the extended and unclasped high affinity conformations; the 9EG7 epitope is marked as red dot at the β1 leg and the FN ligand as beige dimers. ( A ) α5β1 integrin fails to shift from a bent to an extended/unclasped, high affinity state in the absence of talin-1/2 or kindlin-1/2; the bent/clasped conformation brings the EGF-2 domain of the β subunit in close contact with the calf domain of the α5 subunit and prevents exposure of the 9EG7 epitope. ( B ) In the absence of talin (Tln Ko ) and presence of Mn 2+ , kindlin-2 allows adhesion by stabilizing the high affinity conformation of a low number of integrins and the direct binding of paxillin, leading to nucleation of integrins, recruitment of FAK, FAK-dependent signaling and lamellipodia formation. ( C ) In the absence of kindlins (Kind Ko ), talin stabilizes the high affinity conformation of a low number of integrins but does not enable paxillin recruitment and lamellipodia formation. ( D ) In normal fibroblasts, binding of kindlin and talin to the β1 tail is associated with the stabilisation of the unclasped α5β1 heterodimer and 9EG7 epitope exposure. ( E ) Kindlin recruits paxillin and FAK through the kindlin-PH domain and ILK/Pinch/Parvin (IPP; not shown) in a talin-independent manner and induces cell spreading, proliferation and survival. ( F ) The high affinity conformation of α5β1 integrin is stabilized by linkage of the β1 tail to the actin cytoskeleton through talin (and potentially the IPP complex; not shown). The arrow length indicates integrin conformations existing at equilibrium. EGF, epidermal growth factor; FAK, focal adhesion kinase; FN, fibronectin; ILK, integrin-linked kinase; IPP, integrin-linked kinase-Pinch-Parvin; SFK, src family kinases. DOI: http://dx.doi.org/10.7554/eLife.10130.028

    Article Snippet: After two washes in ice-cold PBS to remove unbound antibody, cells were lysed in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM EDTA, and protease inhibitors) and cleared by centrifugation. α5 integrin immuno-complexes were pulled-down by incubation with streptavidin-sepharose (GE Healthcare) overnight at 4°C with gentle agitation.

    Techniques: Binding Assay

    Tln Ko and Kind Ko cells display comparable α5β1 integrin cell surface levels. Live Tln Ctr , Kind Ctr , Tln Ko and Kind Ko cells were incubated with antibodies against α5 integrin (α5) or with an unrelated isotype control (iso) on ice to immunoprecipitate α5 integrin from their cell surface. Following immunoprecipitation, the proteins were analyzed by western blotting to determine the levels of β1 and α5 integrin. DOI: http://dx.doi.org/10.7554/eLife.10130.010

    Journal: eLife

    Article Title: Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

    doi: 10.7554/eLife.10130

    Figure Lengend Snippet: Tln Ko and Kind Ko cells display comparable α5β1 integrin cell surface levels. Live Tln Ctr , Kind Ctr , Tln Ko and Kind Ko cells were incubated with antibodies against α5 integrin (α5) or with an unrelated isotype control (iso) on ice to immunoprecipitate α5 integrin from their cell surface. Following immunoprecipitation, the proteins were analyzed by western blotting to determine the levels of β1 and α5 integrin. DOI: http://dx.doi.org/10.7554/eLife.10130.010

    Article Snippet: After two washes in ice-cold PBS to remove unbound antibody, cells were lysed in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM EDTA, and protease inhibitors) and cleared by centrifugation. α5 integrin immuno-complexes were pulled-down by incubation with streptavidin-sepharose (GE Healthcare) overnight at 4°C with gentle agitation.

    Techniques: Incubation, Immunoprecipitation, Western Blot