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  • 86
    Thermo Fisher recombinant proteins sox9
    CARM1 interacts with and methylates <t>Sox9</t> . (A) Histological analysis of E15.5 wt embryos. Sections of humerus were stained with CARM1 and Col2a1 mRNA probes. Sox9 is stained by ISH and immunohistochemistry (IHC). (B) Sox9 interacts with CARM1 in vitro . Recombinant CARM1 protein and GST-Sox9 fragments were mixed and subjected to a GST-pull down assay. (C) Sox9 recombinant proteins and Histone H3 were incubated with PRMT1 and CARM1 in the presence of [ 3 H] AdoMet. (D) Endogenous Sox9 methylation was detected in mouse primary cultured chondrocytes (left panel). Sox9 multiple point mutants a (mt(a)) and b (mt(b)), wt Sox9, Flag-tagged PABP1 (as a positive control) and Flag-tagged Runx2 (as a negative control) expression plasmids were transfected into 293T cells (right panels). (E) Schematic representation of Sox9 mutants used in the methylation assay.
    Recombinant Proteins Sox9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore recombinant proteins proteins
    Analysis of the RNA Binding Activity of the Full-Length <t>IBV-N</t> Protein and IBV-N29-160 and IBV-N218-329 Fragments The purified IBV-N (lanes 2 and 7), IBV-N29-160 (lanes 3 and 8), IBV-N218-329 (lanes 4 and 9), His-tagged IBV-N29-160 (lanes 5 and 10), and GST (negative control, lanes 6 and 11) were separated on a 15% SDS-PAGE gel. The proteins were either visualized by Coomassie brilliant blue staining (lanes 1–6) or transferred to Hybond C extra membrane (Amersham) and detected by Northwestern blot with a digoxin-labeled RNA probe corresponding to the IBV genome sequence from nucleotides 26,539–27,608 (lanes 7–11). Molecular masses of standard proteins are indicated.
    Recombinant Proteins Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore recombinant proteins recombinant prp16p
    The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the <t>Prp16p-dependent</t> rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .
    Recombinant Proteins Recombinant Prp16p, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems ptx3 recombinant proteins
    The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the <t>Prp16p-dependent</t> rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .
    Ptx3 Recombinant Proteins, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    R&D Systems β1 recombinant proteins
    The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the <t>Prp16p-dependent</t> rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .
    β1 Recombinant Proteins, supplied by R&D Systems, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher e4 recombinant proteins
    The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the <t>Prp16p-dependent</t> rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .
    E4 Recombinant Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc recombinant proteins fgf2
    Feedback activation of FGFR1 signaling leads to adaptive resistance to trametinib in KRAS-mutant lung cancer cells a , Immunoblot analysis of KRAS-mutant lung cancer cell lines H23 and H2030 treated with 25 nM trametinib for various times. b, c, d , qRT-PCR for FGFR1 and <t>FGF2</t> in A549 ( b ), H2030 ( c ) and H460 ( d ) cells treated with trametinib for the indicated times. Data presented as mean normalized for FGFR1 and FGF2 expression ± s.d. (n = 3). e , Immunoblot analysis of A549, H2030, and H358 cells treated with trametinib (25 nM) for various times. f , Quantification of fluorescent cells in competitive proliferation assays in A549, H358, and H460 cells transduced with doxycycline-inducible non-targeting control ( Ren ) or FGFR1 shRNAs. Data presented as mean ± s.d. (n = 3). g , qRT-PCR for FGFR1 in H23 cells transduced with non-targeting control and FGFR1 shRNAs. Data presented as mean normalized for FGFR1 expression ± s.d. (n = 3). h , Quantification of fluorescent cells in competitive proliferation assays in A549 cells transduced with non-targeting control ( Ren ) or the indicated shRNAs. Data presented as mean ± s.d. (n = 3). i , qRT-PCR for FGFR2, FGFR3, and FRS2 in A549 cells transduced with non-targeting control, FGFR2, FGFR3 and FRS2 shRNAs. Data presented as mean normalized for FGFR2, FGFR3, and FRS2 expression ± s.d. (n = 3). b–d , paired two-tailed t -test. f–i , unpaired two-tailed t -test. ns: not significant, * P
    Recombinant Proteins Fgf2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CARM1 interacts with and methylates Sox9 . (A) Histological analysis of E15.5 wt embryos. Sections of humerus were stained with CARM1 and Col2a1 mRNA probes. Sox9 is stained by ISH and immunohistochemistry (IHC). (B) Sox9 interacts with CARM1 in vitro . Recombinant CARM1 protein and GST-Sox9 fragments were mixed and subjected to a GST-pull down assay. (C) Sox9 recombinant proteins and Histone H3 were incubated with PRMT1 and CARM1 in the presence of [ 3 H] AdoMet. (D) Endogenous Sox9 methylation was detected in mouse primary cultured chondrocytes (left panel). Sox9 multiple point mutants a (mt(a)) and b (mt(b)), wt Sox9, Flag-tagged PABP1 (as a positive control) and Flag-tagged Runx2 (as a negative control) expression plasmids were transfected into 293T cells (right panels). (E) Schematic representation of Sox9 mutants used in the methylation assay.

    Journal: BMC Developmental Biology

    Article Title: Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification

    doi: 10.1186/1471-213X-9-47

    Figure Lengend Snippet: CARM1 interacts with and methylates Sox9 . (A) Histological analysis of E15.5 wt embryos. Sections of humerus were stained with CARM1 and Col2a1 mRNA probes. Sox9 is stained by ISH and immunohistochemistry (IHC). (B) Sox9 interacts with CARM1 in vitro . Recombinant CARM1 protein and GST-Sox9 fragments were mixed and subjected to a GST-pull down assay. (C) Sox9 recombinant proteins and Histone H3 were incubated with PRMT1 and CARM1 in the presence of [ 3 H] AdoMet. (D) Endogenous Sox9 methylation was detected in mouse primary cultured chondrocytes (left panel). Sox9 multiple point mutants a (mt(a)) and b (mt(b)), wt Sox9, Flag-tagged PABP1 (as a positive control) and Flag-tagged Runx2 (as a negative control) expression plasmids were transfected into 293T cells (right panels). (E) Schematic representation of Sox9 mutants used in the methylation assay.

    Article Snippet: Purification of Recombinant Proteins Sox9 was synthesized using the BaculoDirect system, according to the manufacturer's protocol (Invitrogen).

    Techniques: Staining, In Situ Hybridization, Immunohistochemistry, In Vitro, Recombinant, Pull Down Assay, Incubation, Methylation, Cell Culture, Positive Control, Negative Control, Expressing, Transfection

    CARM1 regulates Cyclin D1 gene expression . (A) Lysates of SW1353 cells transfected with wt or mutant Flag-Sox9 were immunoprecipitated with anti-Flag antibody. Precipitates were subjected to Western blotting with an anti-beta-catenin (middle panel) and anti-Flag antibody (lower panel), respectively. Interaction of beta-catenin with wt Sox9 was almost abolished in the presense of high-dose CARM1 (upper panel). (B) Left: Expression of Cyclin D1 mRNA (ISH) in the humerus of E16.5 heterozygous, mutant and E14.5 wt and Tg embryos. Right: Expression of Cyclin D1 mRNA from E16.5 limb buds by RT-PCR. (C) Transcriptional regulation of the Cyclin D1 promoter by Flag-Sox9 and pCMX-CARM1 expression plasmids. Activating of Tcf mt reporter was also measured by a luciferase assay. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test (*).

    Journal: BMC Developmental Biology

    Article Title: Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification

    doi: 10.1186/1471-213X-9-47

    Figure Lengend Snippet: CARM1 regulates Cyclin D1 gene expression . (A) Lysates of SW1353 cells transfected with wt or mutant Flag-Sox9 were immunoprecipitated with anti-Flag antibody. Precipitates were subjected to Western blotting with an anti-beta-catenin (middle panel) and anti-Flag antibody (lower panel), respectively. Interaction of beta-catenin with wt Sox9 was almost abolished in the presense of high-dose CARM1 (upper panel). (B) Left: Expression of Cyclin D1 mRNA (ISH) in the humerus of E16.5 heterozygous, mutant and E14.5 wt and Tg embryos. Right: Expression of Cyclin D1 mRNA from E16.5 limb buds by RT-PCR. (C) Transcriptional regulation of the Cyclin D1 promoter by Flag-Sox9 and pCMX-CARM1 expression plasmids. Activating of Tcf mt reporter was also measured by a luciferase assay. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t -test (*).

    Article Snippet: Purification of Recombinant Proteins Sox9 was synthesized using the BaculoDirect system, according to the manufacturer's protocol (Invitrogen).

    Techniques: Expressing, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Luciferase

    CARM1 regulates chondrocytes proliferation . (A) Profiles of primary cultured chondrocytes from Tg mice were evaluated at days 0 and 6. Expression change of Cyclin D1 mRNA during growth of chondrocytes was detected by using RT-PCR. (B) Mouse chondrocyte primary cell cultures were used for for FACS analysis. Cell cycle distribution was determined by propidium iodine staining of nuclear DNA. (C) Model showing functional and physical interactions between Sox9 and beta-catenin regulated by Sox9 methylation in chondrocytes. Interaction between Sox9 and beta-catenin is inhibited by Sox9 methylation caused by CARM1. Inhibition results in chondrocyte proliferation via up-regulation of beta-catenin/Tcf-Lef activity and Cyclin D1 mRNA expression.

    Journal: BMC Developmental Biology

    Article Title: Arginine methyltransferase CARM1/PRMT4 regulates endochondral ossification

    doi: 10.1186/1471-213X-9-47

    Figure Lengend Snippet: CARM1 regulates chondrocytes proliferation . (A) Profiles of primary cultured chondrocytes from Tg mice were evaluated at days 0 and 6. Expression change of Cyclin D1 mRNA during growth of chondrocytes was detected by using RT-PCR. (B) Mouse chondrocyte primary cell cultures were used for for FACS analysis. Cell cycle distribution was determined by propidium iodine staining of nuclear DNA. (C) Model showing functional and physical interactions between Sox9 and beta-catenin regulated by Sox9 methylation in chondrocytes. Interaction between Sox9 and beta-catenin is inhibited by Sox9 methylation caused by CARM1. Inhibition results in chondrocyte proliferation via up-regulation of beta-catenin/Tcf-Lef activity and Cyclin D1 mRNA expression.

    Article Snippet: Purification of Recombinant Proteins Sox9 was synthesized using the BaculoDirect system, according to the manufacturer's protocol (Invitrogen).

    Techniques: Cell Culture, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, FACS, Staining, Functional Assay, Methylation, Inhibition, Activity Assay

    Analysis of the RNA Binding Activity of the Full-Length IBV-N Protein and IBV-N29-160 and IBV-N218-329 Fragments The purified IBV-N (lanes 2 and 7), IBV-N29-160 (lanes 3 and 8), IBV-N218-329 (lanes 4 and 9), His-tagged IBV-N29-160 (lanes 5 and 10), and GST (negative control, lanes 6 and 11) were separated on a 15% SDS-PAGE gel. The proteins were either visualized by Coomassie brilliant blue staining (lanes 1–6) or transferred to Hybond C extra membrane (Amersham) and detected by Northwestern blot with a digoxin-labeled RNA probe corresponding to the IBV genome sequence from nucleotides 26,539–27,608 (lanes 7–11). Molecular masses of standard proteins are indicated.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Analysis of the RNA Binding Activity of the Full-Length IBV-N Protein and IBV-N29-160 and IBV-N218-329 Fragments The purified IBV-N (lanes 2 and 7), IBV-N29-160 (lanes 3 and 8), IBV-N218-329 (lanes 4 and 9), His-tagged IBV-N29-160 (lanes 5 and 10), and GST (negative control, lanes 6 and 11) were separated on a 15% SDS-PAGE gel. The proteins were either visualized by Coomassie brilliant blue staining (lanes 1–6) or transferred to Hybond C extra membrane (Amersham) and detected by Northwestern blot with a digoxin-labeled RNA probe corresponding to the IBV genome sequence from nucleotides 26,539–27,608 (lanes 7–11). Molecular masses of standard proteins are indicated.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques: RNA Binding Assay, Activity Assay, Purification, Negative Control, SDS Page, Staining, Labeling, Sequencing

    Proposed RNA Binding Site of IBV-N (A) Surface representation of the IBV-N29-160 fragment with electrostatic potentials colored in blue (positive) and red (negative). Residues which are suggested to participate in RNA binding are labeled. The N- and C-terminal ends of the polypeptide chains are indicated. (B) Close-up view of the proposed RNA binding site of the IBV-N29-160 fragment. The Cα trace of IBV-N29-160 is displayed. Side chains which are likely to participate in nucleic acid binding are shown as sticks.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Proposed RNA Binding Site of IBV-N (A) Surface representation of the IBV-N29-160 fragment with electrostatic potentials colored in blue (positive) and red (negative). Residues which are suggested to participate in RNA binding are labeled. The N- and C-terminal ends of the polypeptide chains are indicated. (B) Close-up view of the proposed RNA binding site of the IBV-N29-160 fragment. The Cα trace of IBV-N29-160 is displayed. Side chains which are likely to participate in nucleic acid binding are shown as sticks.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques: RNA Binding Assay, Labeling, Binding Assay

    Overall Fold of the IBV-N Protein (A and B) Comparison of the folds adopted by IBV-N29-160 ([A]; shown as a stereoview, top) and the N-terminal domain of the SARS-CoV nucleocapsid protein (B) ( Huang et al., 2004 ). The two proteins are displayed in the same orientation. Secondary structure elements and some residue numbers are indicated. (C) Topology diagram of the IBV-N29-160 protein. Its N- and C-terminal ends are labeled.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Overall Fold of the IBV-N Protein (A and B) Comparison of the folds adopted by IBV-N29-160 ([A]; shown as a stereoview, top) and the N-terminal domain of the SARS-CoV nucleocapsid protein (B) ( Huang et al., 2004 ). The two proteins are displayed in the same orientation. Secondary structure elements and some residue numbers are indicated. (C) Topology diagram of the IBV-N29-160 protein. Its N- and C-terminal ends are labeled.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques: Labeling

    Hypothetical Model for the Assembly of the IBV Ribonucleoprotein Complex (A) Both the N- (cyan) and C-terminal (green) domains of the IBV-N protein can bind RNA (represented as a thin orange line). The basic patch in IBV-N29-160 is depicted by plus signs. Dimerization of the C-terminal domains (arrows) leads to a clustering of IBV-N proteins and to their oligomerization. (B) The endodomain of the integral membrane protein M can provide further contacts to the ribonucleocapsid (see text). However, the precise coupling between RNA recognition and IBV-N multimerization remains uncertain.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Hypothetical Model for the Assembly of the IBV Ribonucleoprotein Complex (A) Both the N- (cyan) and C-terminal (green) domains of the IBV-N protein can bind RNA (represented as a thin orange line). The basic patch in IBV-N29-160 is depicted by plus signs. Dimerization of the C-terminal domains (arrows) leads to a clustering of IBV-N proteins and to their oligomerization. (B) The endodomain of the integral membrane protein M can provide further contacts to the ribonucleocapsid (see text). However, the precise coupling between RNA recognition and IBV-N multimerization remains uncertain.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques:

    Structural Domains of the IBV-N Protein (A) Schematic representation of the IBV-N protein depicting its various domains and clustering of positive charges, as inferred from the present and other studies. (B) SDS-PAGE analysis of the full-length recombinant IBV-N protein of 44.9 kDa (lane 1, arrow) and the N-terminal proteolytically stable fragment of 14.7 kDa spanning residues 29–160 of the sequence which was crystallized (lane 2). The recombinant IBV-N29-160 is shown in lane 3. (C) Typical plate-shaped crystals of the recombinant IBV-N29-160 protein.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Structural Domains of the IBV-N Protein (A) Schematic representation of the IBV-N protein depicting its various domains and clustering of positive charges, as inferred from the present and other studies. (B) SDS-PAGE analysis of the full-length recombinant IBV-N protein of 44.9 kDa (lane 1, arrow) and the N-terminal proteolytically stable fragment of 14.7 kDa spanning residues 29–160 of the sequence which was crystallized (lane 2). The recombinant IBV-N29-160 is shown in lane 3. (C) Typical plate-shaped crystals of the recombinant IBV-N29-160 protein.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques: SDS Page, Recombinant, Sequencing

    Crosslinking Experiments (A) Full-length IBV-N protein. (B) IBV-N29-160 protein, which was crystallized. (C) C-terminal fragment, IBV-N218-329. The nature and concentrations of crosslinking agent are shown. Monomer, dimer, trimer, and tetramer species of the recombinant proteins are indicated.

    Journal: Structure (London, England : 1993)

    Article Title: The Nucleocapsid Protein of Coronavirus Infectious Bronchitis Virus: Crystal Structure of Its N-Terminal Domain and Multimerization Properties

    doi: 10.1016/j.str.2005.08.021

    Figure Lengend Snippet: Crosslinking Experiments (A) Full-length IBV-N protein. (B) IBV-N29-160 protein, which was crystallized. (C) C-terminal fragment, IBV-N218-329. The nature and concentrations of crosslinking agent are shown. Monomer, dimer, trimer, and tetramer species of the recombinant proteins are indicated.

    Article Snippet: Crosslinking Experiments The purified recombinant proteins IBV-N, IBV-N29-160, and IBV-N218-329 were incubated with either glutaraldehyde or SAB (Sigma-Aldrich, St. Louis, MO) for 2 hr at 20°C using a constant amount of protein (5 μg) with increasing amounts of the crosslinking agent.

    Techniques: Recombinant

    Protein-protein interactions among purified clock proteins in vitro . CLOCK, BMAL1, CRY, and PER protein were individually purified from insect cells. Proteins (0.1 n m ) were mixed as indicated, one of the clock proteins was immunoprecipitated and immunoprecipitates were tested for the other interacting proteins by immunoblotting ( IB ). A , PER2 and CRY1 interaction. B , interaction of BMAL1 with CRY1 and PER2. C , interaction of CLOCK with CRY1 and PER2. The input lanes contain 20% of the proteins used in the immunoprecipitation ( IP ) reactions.

    Journal: The Journal of Biological Chemistry

    Article Title: Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock *

    doi: 10.1074/jbc.M111.254680

    Figure Lengend Snippet: Protein-protein interactions among purified clock proteins in vitro . CLOCK, BMAL1, CRY, and PER protein were individually purified from insect cells. Proteins (0.1 n m ) were mixed as indicated, one of the clock proteins was immunoprecipitated and immunoprecipitates were tested for the other interacting proteins by immunoblotting ( IB ). A , PER2 and CRY1 interaction. B , interaction of BMAL1 with CRY1 and PER2. C , interaction of CLOCK with CRY1 and PER2. The input lanes contain 20% of the proteins used in the immunoprecipitation ( IP ) reactions.

    Article Snippet: Pulldown Assay for Protein-Protein Interactions Purified recombinant proteins at 0.1 nm were mixed in 200 μl of binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 100 μg/ml of BSA, 0.05% Nonidet P-40) and incubated at 22 °C for 15 min. Then 20 μl of either FLAG- or V5-agarose (Sigma) beads were added and the mixture was incubated at 4 °C for 90 min. Then, the beads were washed with 1 ml of ice-cold binding buffer 4 times and after the final wash resuspended in 25 μl of SDS loading buffer and heated at 95 °C for 5 min.

    Techniques: Purification, In Vitro, Immunoprecipitation

    The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the Prp16p-dependent rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .

    Journal: Nature structural & molecular biology

    Article Title: Evidence for a group II intron-like catalytic triplex in the spliceosome

    doi: 10.1038/nsmb.2815

    Figure Lengend Snippet: The U6 triplex is present at the branching and exon ligation stages of splicing a, General splicing pathway highlighting the complexes used to probe for the triplex at the catalytic stages. Individual snRNPs are shown as ovals. The relevant complexes are labeled; the dominant negative ATPase mutant that was added to induce accumulation of specific complexes is indicated in parentheses. Note that here we designate as B*(prp16) those spliceosomes that have catalyzed branching but have not undergone the Prp16p-dependent rearrangement. b, Glycerol gradient sedimentation profile of the complexes used for crosslinking. Spliceosomes containing U6- 4S U80 were assembled on Cy3-labeled UBC4 pre-mRNA and then sedimented. For each gradient fraction, the Cy3-labeled pre-mRNA, lariat intermediate, or excised intron, corresponding to B act , B*(prp16), or P complex, respectively, is quantified as a fraction of the total Cy3 signal. ( c,d ) , Denaturing PAGE analysis of radiolabeled U6- 4S U80 recovered after complex isolation, UV irradiation, and immunoprecipitation via Prp16p ( c ) or Prp22p ( d ). The indicated spliceosomal complexes were UV-irradiated and immunoprecipitated after isolation from peak glycerol gradient fractions, as illustrated in b . Representative gels are shown for the glycerol gradient fractionated spliceosomes (GG spliceosome) before (input) and after (αPrp16 or αPrp22) immunoprecipitation. The lower panels in c and d show the UBC4 pre-mRNA substrate present in the fractions used for immunoprecipitation, detected by Cy3. The X1 immunoprecipitation efficiency (relative to X1 present in the input) is quantified in each panel. Error bars represent s.d. from three technical replicates. For full gels see Supplementary Fig. 8f,g,h .

    Article Snippet: Preparation of recombinant proteins Recombinant Prp16p, Prp22p, and Prp2p were expressed in E. coli Rosetta2 DE3pLysS cells (Novagen), essentially as described .

    Techniques: Ligation, Labeling, Dominant Negative Mutation, Mutagenesis, Sedimentation, Activated Clotting Time Assay, Polyacrylamide Gel Electrophoresis, Isolation, Irradiation, Immunoprecipitation

    Feedback activation of FGFR1 signaling leads to adaptive resistance to trametinib in KRAS-mutant lung cancer cells a , Immunoblot analysis of KRAS-mutant lung cancer cell lines H23 and H2030 treated with 25 nM trametinib for various times. b, c, d , qRT-PCR for FGFR1 and FGF2 in A549 ( b ), H2030 ( c ) and H460 ( d ) cells treated with trametinib for the indicated times. Data presented as mean normalized for FGFR1 and FGF2 expression ± s.d. (n = 3). e , Immunoblot analysis of A549, H2030, and H358 cells treated with trametinib (25 nM) for various times. f , Quantification of fluorescent cells in competitive proliferation assays in A549, H358, and H460 cells transduced with doxycycline-inducible non-targeting control ( Ren ) or FGFR1 shRNAs. Data presented as mean ± s.d. (n = 3). g , qRT-PCR for FGFR1 in H23 cells transduced with non-targeting control and FGFR1 shRNAs. Data presented as mean normalized for FGFR1 expression ± s.d. (n = 3). h , Quantification of fluorescent cells in competitive proliferation assays in A549 cells transduced with non-targeting control ( Ren ) or the indicated shRNAs. Data presented as mean ± s.d. (n = 3). i , qRT-PCR for FGFR2, FGFR3, and FRS2 in A549 cells transduced with non-targeting control, FGFR2, FGFR3 and FRS2 shRNAs. Data presented as mean normalized for FGFR2, FGFR3, and FRS2 expression ± s.d. (n = 3). b–d , paired two-tailed t -test. f–i , unpaired two-tailed t -test. ns: not significant, * P

    Journal: Nature

    Article Title: A combinatorial strategy for treating KRAS mutant lung cancer

    doi: 10.1038/nature18600

    Figure Lengend Snippet: Feedback activation of FGFR1 signaling leads to adaptive resistance to trametinib in KRAS-mutant lung cancer cells a , Immunoblot analysis of KRAS-mutant lung cancer cell lines H23 and H2030 treated with 25 nM trametinib for various times. b, c, d , qRT-PCR for FGFR1 and FGF2 in A549 ( b ), H2030 ( c ) and H460 ( d ) cells treated with trametinib for the indicated times. Data presented as mean normalized for FGFR1 and FGF2 expression ± s.d. (n = 3). e , Immunoblot analysis of A549, H2030, and H358 cells treated with trametinib (25 nM) for various times. f , Quantification of fluorescent cells in competitive proliferation assays in A549, H358, and H460 cells transduced with doxycycline-inducible non-targeting control ( Ren ) or FGFR1 shRNAs. Data presented as mean ± s.d. (n = 3). g , qRT-PCR for FGFR1 in H23 cells transduced with non-targeting control and FGFR1 shRNAs. Data presented as mean normalized for FGFR1 expression ± s.d. (n = 3). h , Quantification of fluorescent cells in competitive proliferation assays in A549 cells transduced with non-targeting control ( Ren ) or the indicated shRNAs. Data presented as mean ± s.d. (n = 3). i , qRT-PCR for FGFR2, FGFR3, and FRS2 in A549 cells transduced with non-targeting control, FGFR2, FGFR3 and FRS2 shRNAs. Data presented as mean normalized for FGFR2, FGFR3, and FRS2 expression ± s.d. (n = 3). b–d , paired two-tailed t -test. f–i , unpaired two-tailed t -test. ns: not significant, * P

    Article Snippet: Recombinant proteins FGF2 (8910, Cell Signaling), HGF (100-39, Peprotech), EGF (AF-100-15, Peprotech), and NRG1 (100-03, Peprotech) were used at 50 ngml−1 for 10 minutes.

    Techniques: Activation Assay, Mutagenesis, Quantitative RT-PCR, Expressing, Transduction, Two Tailed Test

    Trametinib in combination with ponatinib synergizes at inhibiting cell proliferation of KRAS-mutant lung cancer cells a , Serum starved H23 (left panel) and 3T3 (right panel) cells were pre-treated with increasing concentration of ponatinib for 24 hr (1, 30, 100, and 300 nM), followed by stimulation with FGF2 (50 ng/ml) for 10 min. Immunoblot analysis for the indicated antibodies is shown. b , Immunoblot analysis of H2030 cells treated with trametinib (25 nM), ponatinib (750 nM), or their combination for the times shown. Cells were pretreated with trametinib for 4 days, followed by co-treatment with ponatinib and trametinib for 2 days. c , Clonogenic assay of H2030, A549, H2009, and H460 cells treated with increasing concentrations of trametinib, ponatinib, or their combination as indicated. d , Percentage of cell growth inhibition at each concentration of trametinib, ponatinib, or their combination in A549, H2009, and H460 cells after is shown. Data presented as mean of three independent experiments (n = 3). e , Combination Index (CI) scores for H23, H2030, A549, H2009, and H460 cells treated with trametinib in combination with ponatinib at the indicated concentrations. Each CI score represents data from at least three independent experiments. For gel source data, see supplementary Fig. 1 . Source data for Extended Data Figure 6.

    Journal: Nature

    Article Title: A combinatorial strategy for treating KRAS mutant lung cancer

    doi: 10.1038/nature18600

    Figure Lengend Snippet: Trametinib in combination with ponatinib synergizes at inhibiting cell proliferation of KRAS-mutant lung cancer cells a , Serum starved H23 (left panel) and 3T3 (right panel) cells were pre-treated with increasing concentration of ponatinib for 24 hr (1, 30, 100, and 300 nM), followed by stimulation with FGF2 (50 ng/ml) for 10 min. Immunoblot analysis for the indicated antibodies is shown. b , Immunoblot analysis of H2030 cells treated with trametinib (25 nM), ponatinib (750 nM), or their combination for the times shown. Cells were pretreated with trametinib for 4 days, followed by co-treatment with ponatinib and trametinib for 2 days. c , Clonogenic assay of H2030, A549, H2009, and H460 cells treated with increasing concentrations of trametinib, ponatinib, or their combination as indicated. d , Percentage of cell growth inhibition at each concentration of trametinib, ponatinib, or their combination in A549, H2009, and H460 cells after is shown. Data presented as mean of three independent experiments (n = 3). e , Combination Index (CI) scores for H23, H2030, A549, H2009, and H460 cells treated with trametinib in combination with ponatinib at the indicated concentrations. Each CI score represents data from at least three independent experiments. For gel source data, see supplementary Fig. 1 . Source data for Extended Data Figure 6.

    Article Snippet: Recombinant proteins FGF2 (8910, Cell Signaling), HGF (100-39, Peprotech), EGF (AF-100-15, Peprotech), and NRG1 (100-03, Peprotech) were used at 50 ngml−1 for 10 minutes.

    Techniques: Mutagenesis, Concentration Assay, Clonogenic Assay, Inhibition

    Feedback activation of FGFR1 mediates adaptive resistance to trametinib in KRAS-mutant lung cells a , qRT-PCR for FGFR1 and FGF2 in H23 cells treated with trametinib for the indicated times (n = 3). b , Immunoblot of H23 cells treated with 25 nM of trametinib for various times. c , Immunoblot of H23 cells transduced with a doxycycline-inducible shRNA targeting FGFR1 and treated with trametinib (25 nM) and doxycycline for the times shown. d , Quantification of fluorescent cells in competitive proliferation assay in H23 and H2030 cells transduced with doxycycline-inducible non-targeting control ( Ren ) or FGFR1 shRNAs (n = 3). e , Clonogenic assay of H23 cells transduced with FGFR1 and non-targeting control shRNAs, and cultured with DMSO or trametinib (25 nM). Relative growth of DMSO- (grey bars) and trametinib-treated cells (blue and red bars) is shown (right) (n = 3). f, g , Quantification of fluorescent cells in competitive proliferation assays in H23 ( f ) and the indicated lung cancer cells ( g ) transduced with doxycycline-inducible non-targeting control ( Ren (Renilla)) or the indicated shRNAs (n = 3). a , Paired two-tailed t -test. d, f, g , Unpaired two-tailed t -test. Data presented as mean ± s.d. ns: not significant, * P

    Journal: Nature

    Article Title: A combinatorial strategy for treating KRAS mutant lung cancer

    doi: 10.1038/nature18600

    Figure Lengend Snippet: Feedback activation of FGFR1 mediates adaptive resistance to trametinib in KRAS-mutant lung cells a , qRT-PCR for FGFR1 and FGF2 in H23 cells treated with trametinib for the indicated times (n = 3). b , Immunoblot of H23 cells treated with 25 nM of trametinib for various times. c , Immunoblot of H23 cells transduced with a doxycycline-inducible shRNA targeting FGFR1 and treated with trametinib (25 nM) and doxycycline for the times shown. d , Quantification of fluorescent cells in competitive proliferation assay in H23 and H2030 cells transduced with doxycycline-inducible non-targeting control ( Ren ) or FGFR1 shRNAs (n = 3). e , Clonogenic assay of H23 cells transduced with FGFR1 and non-targeting control shRNAs, and cultured with DMSO or trametinib (25 nM). Relative growth of DMSO- (grey bars) and trametinib-treated cells (blue and red bars) is shown (right) (n = 3). f, g , Quantification of fluorescent cells in competitive proliferation assays in H23 ( f ) and the indicated lung cancer cells ( g ) transduced with doxycycline-inducible non-targeting control ( Ren (Renilla)) or the indicated shRNAs (n = 3). a , Paired two-tailed t -test. d, f, g , Unpaired two-tailed t -test. Data presented as mean ± s.d. ns: not significant, * P

    Article Snippet: Recombinant proteins FGF2 (8910, Cell Signaling), HGF (100-39, Peprotech), EGF (AF-100-15, Peprotech), and NRG1 (100-03, Peprotech) were used at 50 ngml−1 for 10 minutes.

    Techniques: Activation Assay, Mutagenesis, Quantitative RT-PCR, Transduction, shRNA, Proliferation Assay, Clonogenic Assay, Cell Culture, Two Tailed Test