anti-myc Search Results


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  • 99
    Thermo Fisher anti myc
    Anti Myc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti myc
    Pol V transcripts, small RNA abundance, and <t>DTF1/SHH1</t> enrichment in the clsy1234 mutant. a Pol V transcript levels in the indicated genotypes. b Length distribution of small RNAs in the indicated genotypes. c siRNA abundance at clsy1234 and nrpd1 overlapped hypo-DMRs. d siRNA abundance at clsy1234- specific hypo-DMRs. e siRNA abundance at nrpd1- specific hypo-DMRs. f Relative ChIP signals for DTF1/SHH1 in the indicated genotypes. *Indicates statistical difference between <t>DTF1-MYC/Col-0</t> and DTF1-MYC /clsy1234 according to Student’s t -test ( P
    Anti Myc, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology anti myc
    <t>TERT</t> affects <t>MYC</t> steady-state levels. ( A ) Western blots showing levels of indicated proteins upon knockdown with shControl, shTERT1, shTERT2, shTERT3, and sh Terc and upon reexpression of TERT WT and TERT DN in shTERT3 cells. ( B ) Western blots showing levels of indicated proteins upon TERT WT and TERT DN ectopic expression. TT, TERT+ Terc . ( C ) Gene expression analysis of MYC in P493 cells infected with shControl, shTERT, and sh Terc . # P > 0.05, compared with shControl, Student’s t test. ( D ) Western blot showing steady-state levels of MYC following TERT knockdown in EμMYC B cells using 2 independent shRNAs and in cells with reexpression of human TERT. ( E ) Coimmunoprecipitation between endogenous MYC and TERT in the cells infected with shControl, shTERT, or sh Terc . ( F and G ) ChIP using antibodies against TERT and MYC, followed by qRT-PCR with primers specific for gene promoters containing MYC-binding sites in P493 siControl and siTERT cells. ( H ) Sequential ChIP was performed in P493 cells. ChIP was carried out first with antibody against MYC followed by antibody against either TERT or IgG as indicated. For the Western blots, representative images of n = 3 independent experiments are shown. * P
    Anti Myc, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 6673 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti myc
    <t>TERT</t> affects <t>MYC</t> steady-state levels. ( A ) Western blots showing levels of indicated proteins upon knockdown with shControl, shTERT1, shTERT2, shTERT3, and sh Terc and upon reexpression of TERT WT and TERT DN in shTERT3 cells. ( B ) Western blots showing levels of indicated proteins upon TERT WT and TERT DN ectopic expression. TT, TERT+ Terc . ( C ) Gene expression analysis of MYC in P493 cells infected with shControl, shTERT, and sh Terc . # P > 0.05, compared with shControl, Student’s t test. ( D ) Western blot showing steady-state levels of MYC following TERT knockdown in EμMYC B cells using 2 independent shRNAs and in cells with reexpression of human TERT. ( E ) Coimmunoprecipitation between endogenous MYC and TERT in the cells infected with shControl, shTERT, or sh Terc . ( F and G ) ChIP using antibodies against TERT and MYC, followed by qRT-PCR with primers specific for gene promoters containing MYC-binding sites in P493 siControl and siTERT cells. ( H ) Sequential ChIP was performed in P493 cells. ChIP was carried out first with antibody against MYC followed by antibody against either TERT or IgG as indicated. For the Western blots, representative images of n = 3 independent experiments are shown. * P
    Anti Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc c myc
    <t>TERT</t> affects <t>MYC</t> steady-state levels. ( A ) Western blots showing levels of indicated proteins upon knockdown with shControl, shTERT1, shTERT2, shTERT3, and sh Terc and upon reexpression of TERT WT and TERT DN in shTERT3 cells. ( B ) Western blots showing levels of indicated proteins upon TERT WT and TERT DN ectopic expression. TT, TERT+ Terc . ( C ) Gene expression analysis of MYC in P493 cells infected with shControl, shTERT, and sh Terc . # P > 0.05, compared with shControl, Student’s t test. ( D ) Western blot showing steady-state levels of MYC following TERT knockdown in EμMYC B cells using 2 independent shRNAs and in cells with reexpression of human TERT. ( E ) Coimmunoprecipitation between endogenous MYC and TERT in the cells infected with shControl, shTERT, or sh Terc . ( F and G ) ChIP using antibodies against TERT and MYC, followed by qRT-PCR with primers specific for gene promoters containing MYC-binding sites in P493 siControl and siTERT cells. ( H ) Sequential ChIP was performed in P493 cells. ChIP was carried out first with antibody against MYC followed by antibody against either TERT or IgG as indicated. For the Western blots, representative images of n = 3 independent experiments are shown. * P
    C Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4365 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti c myc antibody
    The N terminus of Rsp3 contains signals for processing and secretion. a Schematic representation of constructs used to analyze N-terminal processing of Rsp3. In all constructs, expression was driven from the constitutive otef promoter and all contain a C-terminal HA-tag. Amino acid sequences from 24 to 70 downstream of the signal peptide (SP) are shown. The position of Myc-tag insertion is depicted in light blue. Amino acids DGGA identified by N-terminal sequencing as N terminus of secreted Rsp3 are indicated in red. The arrow indicates the cleavage site. Amino acids subjected to alanine substitution are indicated in green. b Western blot analysis of Rsp3 secretion. SG200Δrsp3 strains expressing the indicated proteins were grown in CM liquid medium to an OD 600 of 0.6. Proteins from cell pellets and from supernatants (collected after TCA precipitation) were prepared subjected to western blot. The western blots were developed with either anti-HA or anti c-Myc antibodies as indicated. Detection of <t>tubulin</t> via an anti-tubulin antibody served as internal control for a cytosolic protein. c Secretion of Rsp3 variants carrying amino acid substitutions or deletions in the N-terminal domain. SG200Δrsp3 strains expressing the indicated proteins were analyzed by western blot after fractionation in supernatant and cell pellet as in b
    Monoclonal Anti C Myc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore anti myc antibody
    The N terminus of Rsp3 contains signals for processing and secretion. a Schematic representation of constructs used to analyze N-terminal processing of Rsp3. In all constructs, expression was driven from the constitutive otef promoter and all contain a C-terminal HA-tag. Amino acid sequences from 24 to 70 downstream of the signal peptide (SP) are shown. The position of Myc-tag insertion is depicted in light blue. Amino acids DGGA identified by N-terminal sequencing as N terminus of secreted Rsp3 are indicated in red. The arrow indicates the cleavage site. Amino acids subjected to alanine substitution are indicated in green. b Western blot analysis of Rsp3 secretion. SG200Δrsp3 strains expressing the indicated proteins were grown in CM liquid medium to an OD 600 of 0.6. Proteins from cell pellets and from supernatants (collected after TCA precipitation) were prepared subjected to western blot. The western blots were developed with either anti-HA or anti c-Myc antibodies as indicated. Detection of <t>tubulin</t> via an anti-tubulin antibody served as internal control for a cytosolic protein. c Secretion of Rsp3 variants carrying amino acid substitutions or deletions in the N-terminal domain. SG200Δrsp3 strains expressing the indicated proteins were analyzed by western blot after fractionation in supernatant and cell pellet as in b
    Anti Myc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 2226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology mouse anti myc
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Mouse Anti Myc, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti c myc antibody
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Anti C Myc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti c myc
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Anti C Myc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    c myc  (Abcam)
    99
    Abcam c myc
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    C Myc, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti myc
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Anti Myc, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti myc
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Mouse Anti Myc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti myc antibody
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Anti Myc Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti myc antibodies
    <t>BAG2</t> forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and <t>MYC-BAG2</t> were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection
    Anti Myc Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology rabbit anti myc
    Influence of β-TrCP overexpression or suppression on steady-state level and polyubiquitination of CREB-H-ΔTC. ( A ) Immunoblotting of cell lysates derived from HEK293T cells expressing β-TrCP (FLAG-β-T) or β-TrCP-ΔFbox (FLAG-ΔF), ubiquitin (Ub) and CREB-H-ΔTC (ΔTC). ( B ) Immunoblotting of cell lysates derived from HEK293T cells transfected with two independent siRNAs targeting β-TrCP (siβ-T#1 and siβ-T#2). siGFP served as a control. ( C ) Analysis of protein stability by cycloheximide chase assay. HEK293T cells cotransfected with siβ-T#1 and CREB-H-ΔTC plasmid were treated with 200 μM cycloheximide (CHX) for 3, 5 and 8 hours before harvest. Protein samples were analyzed by SDS-PAGE and probed with the indicated antibodies. ( D ) Polyubiquitination analysis. Lysates from HEK293T cells expressing the indicated proteins were immunoprecipitated with <t>anti-V5</t> and the precipitates were analyzed by immunoblotting with <t>anti-MYC,</t> anti-FLAG and anti-V5. The inputs (10%) were probed with anti-V5 and anti-GAPDH.
    Rabbit Anti Myc, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 954 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covance anti myc
    The interactions of mlc1 mutant proteins with Myo2p, Iqg1p, and <t>Myo1p.</t> (A) <t>Myo2p-MYC</t> was precipitated with anti-MYC-agarose beads from cell lysates of strains YJL176A ( MYO1:HA MYO2:MYC ) carrying pUG34 (vector), pUG34-MLC1 (Mlc1p), pUG34-MLC1-93 (mlc1-93p), or pUG34-MLC1-11 (mlc1-11p), respectively. Myo2p-MYC, Mlc1p-GFP, and its derivatives in the bound and in the input fractions were detected by Western-blot analysis with antibodies against MYC or GFP as indicated in A. (B) As in A except that strain YJL175A ( myo1 Δ IQG1:HA MYO2:MYC ) carrying the different plasmids and the anti-HA-agarose beads were used to do the pull down. (C) As in A except that anti-HA-agarose beads were used to do the pull down. (D) Summary of the interactions between Mlc1p derivatives and its binding partners. (E) Position of the mlc1-11 and mlc1-93 mutations on the three-dimensional structure of the IQ2 (from Myo2p)–Mlc1p complex ( Terrak et al., 2003 ).
    Anti Myc, supplied by Covance, used in various techniques. Bioz Stars score: 93/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti c myc antibody y69
    The interactions of mlc1 mutant proteins with Myo2p, Iqg1p, and <t>Myo1p.</t> (A) <t>Myo2p-MYC</t> was precipitated with anti-MYC-agarose beads from cell lysates of strains YJL176A ( MYO1:HA MYO2:MYC ) carrying pUG34 (vector), pUG34-MLC1 (Mlc1p), pUG34-MLC1-93 (mlc1-93p), or pUG34-MLC1-11 (mlc1-11p), respectively. Myo2p-MYC, Mlc1p-GFP, and its derivatives in the bound and in the input fractions were detected by Western-blot analysis with antibodies against MYC or GFP as indicated in A. (B) As in A except that strain YJL175A ( myo1 Δ IQG1:HA MYO2:MYC ) carrying the different plasmids and the anti-HA-agarose beads were used to do the pull down. (C) As in A except that anti-HA-agarose beads were used to do the pull down. (D) Summary of the interactions between Mlc1p derivatives and its binding partners. (E) Position of the mlc1-11 and mlc1-93 mutations on the three-dimensional structure of the IQ2 (from Myo2p)–Mlc1p complex ( Terrak et al., 2003 ).
    Anti C Myc Antibody Y69, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti c myc agarose affinity gel antibody
    The interactions of mlc1 mutant proteins with Myo2p, Iqg1p, and <t>Myo1p.</t> (A) <t>Myo2p-MYC</t> was precipitated with anti-MYC-agarose beads from cell lysates of strains YJL176A ( MYO1:HA MYO2:MYC ) carrying pUG34 (vector), pUG34-MLC1 (Mlc1p), pUG34-MLC1-93 (mlc1-93p), or pUG34-MLC1-11 (mlc1-11p), respectively. Myo2p-MYC, Mlc1p-GFP, and its derivatives in the bound and in the input fractions were detected by Western-blot analysis with antibodies against MYC or GFP as indicated in A. (B) As in A except that strain YJL175A ( myo1 Δ IQG1:HA MYO2:MYC ) carrying the different plasmids and the anti-HA-agarose beads were used to do the pull down. (C) As in A except that anti-HA-agarose beads were used to do the pull down. (D) Summary of the interactions between Mlc1p derivatives and its binding partners. (E) Position of the mlc1-11 and mlc1-93 mutations on the three-dimensional structure of the IQ2 (from Myo2p)–Mlc1p complex ( Terrak et al., 2003 ).
    Anti C Myc Agarose Affinity Gel Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1320 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pol V transcripts, small RNA abundance, and DTF1/SHH1 enrichment in the clsy1234 mutant. a Pol V transcript levels in the indicated genotypes. b Length distribution of small RNAs in the indicated genotypes. c siRNA abundance at clsy1234 and nrpd1 overlapped hypo-DMRs. d siRNA abundance at clsy1234- specific hypo-DMRs. e siRNA abundance at nrpd1- specific hypo-DMRs. f Relative ChIP signals for DTF1/SHH1 in the indicated genotypes. *Indicates statistical difference between DTF1-MYC/Col-0 and DTF1-MYC /clsy1234 according to Student’s t -test ( P

    Journal: Cell Discovery

    Article Title: Four putative SWI2/SNF2 chromatin remodelers have dual roles in regulating DNA methylation in Arabidopsis

    doi: 10.1038/s41421-018-0056-8

    Figure Lengend Snippet: Pol V transcripts, small RNA abundance, and DTF1/SHH1 enrichment in the clsy1234 mutant. a Pol V transcript levels in the indicated genotypes. b Length distribution of small RNAs in the indicated genotypes. c siRNA abundance at clsy1234 and nrpd1 overlapped hypo-DMRs. d siRNA abundance at clsy1234- specific hypo-DMRs. e siRNA abundance at nrpd1- specific hypo-DMRs. f Relative ChIP signals for DTF1/SHH1 in the indicated genotypes. *Indicates statistical difference between DTF1-MYC/Col-0 and DTF1-MYC /clsy1234 according to Student’s t -test ( P

    Article Snippet: The antibody used for DTF1 ChIP assays was anti-MYC (16–219, Millipore).

    Techniques: Mutagenesis, Chromatin Immunoprecipitation

    TERT affects MYC steady-state levels. ( A ) Western blots showing levels of indicated proteins upon knockdown with shControl, shTERT1, shTERT2, shTERT3, and sh Terc and upon reexpression of TERT WT and TERT DN in shTERT3 cells. ( B ) Western blots showing levels of indicated proteins upon TERT WT and TERT DN ectopic expression. TT, TERT+ Terc . ( C ) Gene expression analysis of MYC in P493 cells infected with shControl, shTERT, and sh Terc . # P > 0.05, compared with shControl, Student’s t test. ( D ) Western blot showing steady-state levels of MYC following TERT knockdown in EμMYC B cells using 2 independent shRNAs and in cells with reexpression of human TERT. ( E ) Coimmunoprecipitation between endogenous MYC and TERT in the cells infected with shControl, shTERT, or sh Terc . ( F and G ) ChIP using antibodies against TERT and MYC, followed by qRT-PCR with primers specific for gene promoters containing MYC-binding sites in P493 siControl and siTERT cells. ( H ) Sequential ChIP was performed in P493 cells. ChIP was carried out first with antibody against MYC followed by antibody against either TERT or IgG as indicated. For the Western blots, representative images of n = 3 independent experiments are shown. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: TERT affects MYC steady-state levels. ( A ) Western blots showing levels of indicated proteins upon knockdown with shControl, shTERT1, shTERT2, shTERT3, and sh Terc and upon reexpression of TERT WT and TERT DN in shTERT3 cells. ( B ) Western blots showing levels of indicated proteins upon TERT WT and TERT DN ectopic expression. TT, TERT+ Terc . ( C ) Gene expression analysis of MYC in P493 cells infected with shControl, shTERT, and sh Terc . # P > 0.05, compared with shControl, Student’s t test. ( D ) Western blot showing steady-state levels of MYC following TERT knockdown in EμMYC B cells using 2 independent shRNAs and in cells with reexpression of human TERT. ( E ) Coimmunoprecipitation between endogenous MYC and TERT in the cells infected with shControl, shTERT, or sh Terc . ( F and G ) ChIP using antibodies against TERT and MYC, followed by qRT-PCR with primers specific for gene promoters containing MYC-binding sites in P493 siControl and siTERT cells. ( H ) Sequential ChIP was performed in P493 cells. ChIP was carried out first with antibody against MYC followed by antibody against either TERT or IgG as indicated. For the Western blots, representative images of n = 3 independent experiments are shown. * P

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: Western Blot, Expressing, Infection, Chromatin Immunoprecipitation, Quantitative RT-PCR, Binding Assay

    Graphical model. TERT levels, which limit the reconstitution of telomerase activity in normal cells, are upregulated by increased MYC in cancer cells. TERT, in turn, enhances MYC stability and function, thereby regulating its own levels and telomerase activity. This function of TERT does not require Terc and is independent of its function at telomeres. Enhanced MYC stability and function in high-TERT and high-MYC cells, a consequence of this feed-forward mechanism, lead to enhanced oncogenesis due to downstream targets of MYC, which are known to regulate aspects of cell cycle, proliferation, and metabolism.

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: Graphical model. TERT levels, which limit the reconstitution of telomerase activity in normal cells, are upregulated by increased MYC in cancer cells. TERT, in turn, enhances MYC stability and function, thereby regulating its own levels and telomerase activity. This function of TERT does not require Terc and is independent of its function at telomeres. Enhanced MYC stability and function in high-TERT and high-MYC cells, a consequence of this feed-forward mechanism, lead to enhanced oncogenesis due to downstream targets of MYC, which are known to regulate aspects of cell cycle, proliferation, and metabolism.

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: Activity Assay

    Homozygous deletion of Tert delays MYC-driven lymphomagenesis in vivo. ( A ) Kaplan-Meier curves showing the tumor-free survival of EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– mice. ( B ) Kaplan-Meier curves showing the tumor-free survival of EμMYC Terc +/+ , EμMYC Terc +/– , and EμMYC Terc –/– mice. ( C ) MYC levels in spleen B cells from 12-week-old EμMYC Tert +/+ ( n = 3), EμMYC Tert +/– ( n = 4), and EμMYC Tert –/– mice ( n = 5). ( D ) wbc counts, spleen weights, and tumor weights in 12-week-old EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– mice. Each dot represents an independent mouse. The horizontal bars represent the mean. ( E ) Representative immunohistochemistry images (original magnification, ×10), showing B220 and Ki67 staining in EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– spleens. Spleens from at least 3 mice of each genotype were analyzed. One-way ANOVA with Dunnett’s multiple comparison test was used to compare differences between the various genotypes and EμMYC Tert +/+ . P

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: Homozygous deletion of Tert delays MYC-driven lymphomagenesis in vivo. ( A ) Kaplan-Meier curves showing the tumor-free survival of EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– mice. ( B ) Kaplan-Meier curves showing the tumor-free survival of EμMYC Terc +/+ , EμMYC Terc +/– , and EμMYC Terc –/– mice. ( C ) MYC levels in spleen B cells from 12-week-old EμMYC Tert +/+ ( n = 3), EμMYC Tert +/– ( n = 4), and EμMYC Tert –/– mice ( n = 5). ( D ) wbc counts, spleen weights, and tumor weights in 12-week-old EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– mice. Each dot represents an independent mouse. The horizontal bars represent the mean. ( E ) Representative immunohistochemistry images (original magnification, ×10), showing B220 and Ki67 staining in EμMYC Tert +/+ , EμMYC Tert +/– , and EμMYC Tert –/– spleens. Spleens from at least 3 mice of each genotype were analyzed. One-way ANOVA with Dunnett’s multiple comparison test was used to compare differences between the various genotypes and EμMYC Tert +/+ . P

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: In Vivo, Mouse Assay, Immunohistochemistry, Staining

    Genome-wide effect of TERT on MYC binding to target promoters and expression of MYC-regulated target genes. MYC ChIP-Seq was performed in ( C ) P493 shControl, shTERT1, and sh Terc or EμMYC Tert +/+ and EμMYC Tert –/– cells. ( A and B ) Peak profile centering on the transcriptional start site. Peaks were normalized to a width of 300 bp, and the frequency of binding events (1 equals all events) was calculated at each distance to the transcriptional start site. ( C and E ) Read density profile plots focusing on the transcriptional start site of MYC-bound promoters. Read densities at all promoters bound by MYC within 500 bp around the transcriptional start site in ( C ) P493 shControl, shTERT1, and sh Terc or ( E ) EμMYC Tert +/+ and EμMYC Tert –/– cells were calculated and plotted as a heat map of normalized, background subtracted reads per sample. The black line indicates the transcriptional start site. ( D and F ) Representative examples of MYC binding to selected promoters. RPM, reads per million. ( G ) Transcriptional outcome of altered MYC binding upon loss of TERT. Bound and differentially expressed promoters were identified in P493 shControl and P493 shTERT1 cells. The change in MYC binding in shTERT1 is indicated on the x axis as the log2-fold change (normalized, background subtracted read densities within 500 bp upstream and downstream of the transcriptional start site around the transcriptional start site). Changes in expression are represented as normalized log2-fold change values of the respective genes on the y axis. The blue line is a linear smooth with a 0.95 confidence interval, indicating the general trend of the data set. ( H ) Enriched GO categories of bound and differentially expressed promoters, as indicated in G . The red line indicates a q value of 0.01, as reported by DAVID.

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: Genome-wide effect of TERT on MYC binding to target promoters and expression of MYC-regulated target genes. MYC ChIP-Seq was performed in ( C ) P493 shControl, shTERT1, and sh Terc or EμMYC Tert +/+ and EμMYC Tert –/– cells. ( A and B ) Peak profile centering on the transcriptional start site. Peaks were normalized to a width of 300 bp, and the frequency of binding events (1 equals all events) was calculated at each distance to the transcriptional start site. ( C and E ) Read density profile plots focusing on the transcriptional start site of MYC-bound promoters. Read densities at all promoters bound by MYC within 500 bp around the transcriptional start site in ( C ) P493 shControl, shTERT1, and sh Terc or ( E ) EμMYC Tert +/+ and EμMYC Tert –/– cells were calculated and plotted as a heat map of normalized, background subtracted reads per sample. The black line indicates the transcriptional start site. ( D and F ) Representative examples of MYC binding to selected promoters. RPM, reads per million. ( G ) Transcriptional outcome of altered MYC binding upon loss of TERT. Bound and differentially expressed promoters were identified in P493 shControl and P493 shTERT1 cells. The change in MYC binding in shTERT1 is indicated on the x axis as the log2-fold change (normalized, background subtracted read densities within 500 bp upstream and downstream of the transcriptional start site around the transcriptional start site). Changes in expression are represented as normalized log2-fold change values of the respective genes on the y axis. The blue line is a linear smooth with a 0.95 confidence interval, indicating the general trend of the data set. ( H ) Enriched GO categories of bound and differentially expressed promoters, as indicated in G . The red line indicates a q value of 0.01, as reported by DAVID.

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: Genome Wide, Binding Assay, Expressing, Chromatin Immunoprecipitation

    Phenotypic effects of TERT inhibition or depletion on MYC-dependent processes. ( A ) Gene expression analysis of the indicated genes in P493 cells infected with vector, shTERT1, and shTERT1 + MYC ( n = 3). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: Phenotypic effects of TERT inhibition or depletion on MYC-dependent processes. ( A ) Gene expression analysis of the indicated genes in P493 cells infected with vector, shTERT1, and shTERT1 + MYC ( n = 3). ** P

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: Inhibition, Expressing, Infection, Plasmid Preparation

    TERT regulates MYC half-life and ubiquitination. ( A and B ) Western blots showing MYC levels upon TERT or Terc knockdown following cycloheximide (CHX) treatment for indicated time points in P493 cells and EμMYC B cells. UT, untreated. ( C and D ) Western blots in P493 and EμMYC B cells showing the levels of MYC, TERT, and actin upon TERT knockdown, with and without MG132 treatment (10 μM, 2 hours). ( E ) Western blots showing levels of indicated proteins upon vector, TERT WT, and TERT DN expression. ( F ) Coimmunoprecipitation between endogenous MYC and GSK3β in cells infected with shRNA against control (–) or TERT (+) and immunoblotting with indicated antibodies. ( G ) 293T cells were transfected with HA-MYC and His-ubiquitin, along with shControl or shTERT1. 48 hours after transfection cells were harvested, immunoprecipitated with Ni-NTA beads, and Western blotted with HA. ( H ) In vivo MYC ubiquitination assay in cells transfected with control or TERT shRNAs, along with HA-ubiquitin. Immunoprecipitation and immunoblotting from lysates or immunoprecipitated material were performed by using indicated antibodies. For the Western blots, representative images of n = 3 independent experiments are shown.

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: TERT regulates MYC half-life and ubiquitination. ( A and B ) Western blots showing MYC levels upon TERT or Terc knockdown following cycloheximide (CHX) treatment for indicated time points in P493 cells and EμMYC B cells. UT, untreated. ( C and D ) Western blots in P493 and EμMYC B cells showing the levels of MYC, TERT, and actin upon TERT knockdown, with and without MG132 treatment (10 μM, 2 hours). ( E ) Western blots showing levels of indicated proteins upon vector, TERT WT, and TERT DN expression. ( F ) Coimmunoprecipitation between endogenous MYC and GSK3β in cells infected with shRNA against control (–) or TERT (+) and immunoblotting with indicated antibodies. ( G ) 293T cells were transfected with HA-MYC and His-ubiquitin, along with shControl or shTERT1. 48 hours after transfection cells were harvested, immunoprecipitated with Ni-NTA beads, and Western blotted with HA. ( H ) In vivo MYC ubiquitination assay in cells transfected with control or TERT shRNAs, along with HA-ubiquitin. Immunoprecipitation and immunoblotting from lysates or immunoprecipitated material were performed by using indicated antibodies. For the Western blots, representative images of n = 3 independent experiments are shown.

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: Western Blot, Plasmid Preparation, Expressing, Infection, shRNA, Transfection, Immunoprecipitation, In Vivo, Ubiquitin Assay

    Effect of acute depletion of TERT on MYC-driven lymphomas in vivo. ( A ) Western blots showing levels of TERT, MYC, and actin in different tissues of EμMYC mice. ( B ) Kaplan-Meier survival analysis for mice xenografted with shControl, shTERT-A, shTERT-B, ectopic TERT, shTERT-A + ectopic TERT, shTERT-B + ectopic TERT, or sh Terc EμMYC primary lymphoma cells. P

    Journal: The Journal of Clinical Investigation

    Article Title: Telomerase regulates MYC-driven oncogenesis independent of its reverse transcriptase activity

    doi: 10.1172/JCI79134

    Figure Lengend Snippet: Effect of acute depletion of TERT on MYC-driven lymphomas in vivo. ( A ) Western blots showing levels of TERT, MYC, and actin in different tissues of EμMYC mice. ( B ) Kaplan-Meier survival analysis for mice xenografted with shControl, shTERT-A, shTERT-B, ectopic TERT, shTERT-A + ectopic TERT, shTERT-B + ectopic TERT, or sh Terc EμMYC primary lymphoma cells. P

    Article Snippet: The antibodies used for Western blots are as follows: anti-TERT (Epitomics); anti-MYC (sc-764; sc-40; A-14) and anti-MAX (sc-197) (Santa Cruz Biotechnology); anti-MYC (phospho S62) and anti-MYC (phospho T58) (Abcam); anti-GSK3β (BD Biosciences); and anti–β-actin (Sigma-Aldrich) (see complete unedited blots in the supplemental material).

    Techniques: In Vivo, Western Blot, Mouse Assay

    Inhibitors of PKC and p38 specifically suppress the TPA-mediated expression levels of c- myc , p38, and ZEBRA. (A) GT38 cells were pretreated with a PKC inhibitor H7 (100 μM) or staurosporine (St; 0.1 μM) for 1 h, and then exposed to TPA (20 ng/ml) for 4 h. Total RNAs were extracted and analyzed for c- myc mRNA expression by Northern blotting. After stripping, the membrane was reprobed with GAPDH. (B) GT38 cells were pretreated with H7 or staurosporine (St) or SB203580 (SB; 10 μM) for 1 h and then treated with TPA. Cell lysates were extracted for p38, c-Myc, and ZEBRA at 0.5, 4, and 48 h, respectively, after TPA treatment. The phosphorylation of p38, total p38 (t-p38), c-Myc, phospho-c-Myc, ZEBRA, and β-actin were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, anti-phospho-c-Myc, anti-ZEBRA, or anti-β-actin antibodies. The relative densitometric units of c- myc to GAPDH (panel A) and of phospho-p38 to total p38, as well as c-Myc, phospho-c-Myc, and ZEBRA to β-actin (panel B) were quantified with a phosphorimage system. The data represent three independent experiments and the standard deviations are shown.

    Journal: Journal of Virology

    Article Title: Inhibition of Epstein-Barr Virus (EBV) Reactivation by Short Interfering RNAs Targeting p38 Mitogen-Activated Protein Kinase or c-myc in EBV-Positive Epithelial Cells

    doi: 10.1128/JVI.78.21.11798-11806.2004

    Figure Lengend Snippet: Inhibitors of PKC and p38 specifically suppress the TPA-mediated expression levels of c- myc , p38, and ZEBRA. (A) GT38 cells were pretreated with a PKC inhibitor H7 (100 μM) or staurosporine (St; 0.1 μM) for 1 h, and then exposed to TPA (20 ng/ml) for 4 h. Total RNAs were extracted and analyzed for c- myc mRNA expression by Northern blotting. After stripping, the membrane was reprobed with GAPDH. (B) GT38 cells were pretreated with H7 or staurosporine (St) or SB203580 (SB; 10 μM) for 1 h and then treated with TPA. Cell lysates were extracted for p38, c-Myc, and ZEBRA at 0.5, 4, and 48 h, respectively, after TPA treatment. The phosphorylation of p38, total p38 (t-p38), c-Myc, phospho-c-Myc, ZEBRA, and β-actin were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, anti-phospho-c-Myc, anti-ZEBRA, or anti-β-actin antibodies. The relative densitometric units of c- myc to GAPDH (panel A) and of phospho-p38 to total p38, as well as c-Myc, phospho-c-Myc, and ZEBRA to β-actin (panel B) were quantified with a phosphorimage system. The data represent three independent experiments and the standard deviations are shown.

    Article Snippet: Immunoblottings were performed by using the following antibodies: a 1:100 dilution of anti-c-Myc and a 1:200 dilution of anti-β-actin (Santa Cruz Biotechnology, Inc., Calif.); a 1:1,000 dilution of anti-phospho-c-Myc, anti-phospho-p38, and anti-p38 (New England, Biolabs Inc., Beverly, Mass.); and a 1:100 dilution of anti-ZEBRA monoclonal antibodies (developed in our laboratory).

    Techniques: Expressing, Northern Blot, Stripping Membranes, Western Blot

    TPA induces c- myc and p38 gene expression and EBV reactivation. GT38 cells were incubated with TPA (20 ng/ml). (A) Total RNAs were isolated from the cells at indicated times, and the expression levels of BZLF1, BRLF1, and c- myc genes were analyzed by Northern blotting. GAPDH was analyzed as an internal control standard. (B) Cell lysates were isolated at indicated times, and the phosphorylation of p38, total p38 (t-p38), c-Myc, and ZEBRA were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, and anti-ZEBRA antibodies. Total p38 and β-actin were analyzed as internal control standards. The relative densitometric units of BRLF1, BZLF1, and c- myc to GAPDH (panel A) and of phospho-p38 to total p38 and c-Myc and ZEBRA to β-actin (panel B) were quantified by a phosphorimage system. The data represent three independent experiments, and the standard deviations are shown.

    Journal: Journal of Virology

    Article Title: Inhibition of Epstein-Barr Virus (EBV) Reactivation by Short Interfering RNAs Targeting p38 Mitogen-Activated Protein Kinase or c-myc in EBV-Positive Epithelial Cells

    doi: 10.1128/JVI.78.21.11798-11806.2004

    Figure Lengend Snippet: TPA induces c- myc and p38 gene expression and EBV reactivation. GT38 cells were incubated with TPA (20 ng/ml). (A) Total RNAs were isolated from the cells at indicated times, and the expression levels of BZLF1, BRLF1, and c- myc genes were analyzed by Northern blotting. GAPDH was analyzed as an internal control standard. (B) Cell lysates were isolated at indicated times, and the phosphorylation of p38, total p38 (t-p38), c-Myc, and ZEBRA were analyzed by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc, and anti-ZEBRA antibodies. Total p38 and β-actin were analyzed as internal control standards. The relative densitometric units of BRLF1, BZLF1, and c- myc to GAPDH (panel A) and of phospho-p38 to total p38 and c-Myc and ZEBRA to β-actin (panel B) were quantified by a phosphorimage system. The data represent three independent experiments, and the standard deviations are shown.

    Article Snippet: Immunoblottings were performed by using the following antibodies: a 1:100 dilution of anti-c-Myc and a 1:200 dilution of anti-β-actin (Santa Cruz Biotechnology, Inc., Calif.); a 1:1,000 dilution of anti-phospho-c-Myc, anti-phospho-p38, and anti-p38 (New England, Biolabs Inc., Beverly, Mass.); and a 1:100 dilution of anti-ZEBRA monoclonal antibodies (developed in our laboratory).

    Techniques: Expressing, Incubation, Isolation, Northern Blot, Western Blot

    TPA-induced phosphorylation of p38 and activation of c-Myc were inhibited by the NO donor SNAP. GT38 cells were pretreated with SNAP at indicated concentrations for 1 h prior to TPA (20 ng/ml) treatment. Cell lysates were prepared for phosphorylation of p38 and total p38 (t-p38) and of c-Myc at 0.5 and 4 h, respectively, after TPA treatment. Cell lysates were separated by SDS-PAGE and blotted onto membranes. Phosphorylated p38, p38, and c-Myc were detected by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc or anti-β-actin antibodies. The p38 and β-actin were used as internal standards. The relative densitometric units of phosphorylation of p38 and activation of c-Myc were determined to p38 and β-actin signals, respectively. The data represent three independent experiments and the standard deviations are shown.

    Journal: Journal of Virology

    Article Title: Inhibition of Epstein-Barr Virus (EBV) Reactivation by Short Interfering RNAs Targeting p38 Mitogen-Activated Protein Kinase or c-myc in EBV-Positive Epithelial Cells

    doi: 10.1128/JVI.78.21.11798-11806.2004

    Figure Lengend Snippet: TPA-induced phosphorylation of p38 and activation of c-Myc were inhibited by the NO donor SNAP. GT38 cells were pretreated with SNAP at indicated concentrations for 1 h prior to TPA (20 ng/ml) treatment. Cell lysates were prepared for phosphorylation of p38 and total p38 (t-p38) and of c-Myc at 0.5 and 4 h, respectively, after TPA treatment. Cell lysates were separated by SDS-PAGE and blotted onto membranes. Phosphorylated p38, p38, and c-Myc were detected by Western blotting with anti-phospho-p38, anti-p38, anti-c-Myc or anti-β-actin antibodies. The p38 and β-actin were used as internal standards. The relative densitometric units of phosphorylation of p38 and activation of c-Myc were determined to p38 and β-actin signals, respectively. The data represent three independent experiments and the standard deviations are shown.

    Article Snippet: Immunoblottings were performed by using the following antibodies: a 1:100 dilution of anti-c-Myc and a 1:200 dilution of anti-β-actin (Santa Cruz Biotechnology, Inc., Calif.); a 1:1,000 dilution of anti-phospho-c-Myc, anti-phospho-p38, and anti-p38 (New England, Biolabs Inc., Beverly, Mass.); and a 1:100 dilution of anti-ZEBRA monoclonal antibodies (developed in our laboratory).

    Techniques: Activation Assay, SDS Page, Western Blot

    siRNAs targeting p38 and c- myc specifically inhibit the expression of respective genes and ZEBRA expression. (A) GT38 cells were transfected with p38 siRNA (0.1 μM) or scrambled siRNA (scr) and incubated for 48 and 72 h or for 72 h and then treated with TPA for 30 min for phosphorylated p38 and total p38 (t-p38) and 48 h for ZEBRA. The cell extracts were analyzed for phosphorylation of p38, total p38, ZEBRA, and β-actin by Western blotting. (B) GT38 cells were transfected with siRNA (0.1 μM) targeted to c- myc for 48 or 72 h or with scrambled (scr) siRNA for 72 h and then treated with TPA (20 ng/ml) for 4 or 48 h for the expression analysis of c-Myc and ZEBRA, respectively. Cell lysates were harvested from siRNA-transfected or untransfected GT38 cells at indicated times posttransfection. C-Myc and ZEBRA expression were analyzed by Western blotting by using anti-c-Myc, anti-ZEBRA, and anti-β-actin antibodies. The signals of phosphor-p38, t-p38, and ZEBRA (panel A) and of c-Myc and ZEBRA (panel B) were quantified by a phosphorimage system. The relative protein level was measured as ratio of β-actin protein. The data represent three independent experiments, and the standard deviations are shown.

    Journal: Journal of Virology

    Article Title: Inhibition of Epstein-Barr Virus (EBV) Reactivation by Short Interfering RNAs Targeting p38 Mitogen-Activated Protein Kinase or c-myc in EBV-Positive Epithelial Cells

    doi: 10.1128/JVI.78.21.11798-11806.2004

    Figure Lengend Snippet: siRNAs targeting p38 and c- myc specifically inhibit the expression of respective genes and ZEBRA expression. (A) GT38 cells were transfected with p38 siRNA (0.1 μM) or scrambled siRNA (scr) and incubated for 48 and 72 h or for 72 h and then treated with TPA for 30 min for phosphorylated p38 and total p38 (t-p38) and 48 h for ZEBRA. The cell extracts were analyzed for phosphorylation of p38, total p38, ZEBRA, and β-actin by Western blotting. (B) GT38 cells were transfected with siRNA (0.1 μM) targeted to c- myc for 48 or 72 h or with scrambled (scr) siRNA for 72 h and then treated with TPA (20 ng/ml) for 4 or 48 h for the expression analysis of c-Myc and ZEBRA, respectively. Cell lysates were harvested from siRNA-transfected or untransfected GT38 cells at indicated times posttransfection. C-Myc and ZEBRA expression were analyzed by Western blotting by using anti-c-Myc, anti-ZEBRA, and anti-β-actin antibodies. The signals of phosphor-p38, t-p38, and ZEBRA (panel A) and of c-Myc and ZEBRA (panel B) were quantified by a phosphorimage system. The relative protein level was measured as ratio of β-actin protein. The data represent three independent experiments, and the standard deviations are shown.

    Article Snippet: Immunoblottings were performed by using the following antibodies: a 1:100 dilution of anti-c-Myc and a 1:200 dilution of anti-β-actin (Santa Cruz Biotechnology, Inc., Calif.); a 1:1,000 dilution of anti-phospho-c-Myc, anti-phospho-p38, and anti-p38 (New England, Biolabs Inc., Beverly, Mass.); and a 1:100 dilution of anti-ZEBRA monoclonal antibodies (developed in our laboratory).

    Techniques: Expressing, Transfection, Incubation, Western Blot

    MYC hyperactivation is synthetic lethal with XBP1 inhibition. ( A ) Clonogenic growth of MCF10A MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ . ( B ) Immunoblot of MYC-ER in nuclear extracts of MCF10A MYC-ER cells treated with 4-OHT for 24 hours. ( C ) Chemical structure of 8866. ( D ) XBP1 -splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. ( E ) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. ( F ) Schematic diagram of fluorescence-based RNA cleavage assay. ( G ) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. ( H ) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. ( I ) Clonogenic growth of MCF10A MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A MYC-ER –GFP cells. In A and I , data are presented as mean ± SD of biological triplicates. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

    doi: 10.1172/JCI95873

    Figure Lengend Snippet: MYC hyperactivation is synthetic lethal with XBP1 inhibition. ( A ) Clonogenic growth of MCF10A MYC-ER cells transduced with shRNAs against XBP1 or LacZ and treated with different doses of 4-OHT. Ethanol was used as vehicle for 4-OHT. Changes in colony number were compared with vehicle-treated cells expressing shLacZ . ( B ) Immunoblot of MYC-ER in nuclear extracts of MCF10A MYC-ER cells treated with 4-OHT for 24 hours. ( C ) Chemical structure of 8866. ( D ) XBP1 -splicing assay in 293T cells that were treated with different doses of 8866 in the presence of DMSO or 5 μg/ml TM for 6 hours. ( E ) SUM159 cells were treated with DMSO or 5 μM 8866 in the presence of 5 μg/ml TM for 6 hours. ChIP assays were performed using anti-XBP1s antibody. Data are presented relative to input and shown as mean ± SD of technical triplicates. ( F ) Schematic diagram of fluorescence-based RNA cleavage assay. ( G ) Cytosolic portions of IRE1 protein or RNase A were incubated with hairpin XBP1 RNA substrate in the presence of various doses of 8866. Cleavage reactions were monitored by fluorescence intensity. ( H ) Immunoblot of IRE1 phosphorylation (phos-tag SDS-PAGE), ATF6 cleavage (ATF6p), PERK, and eIF2α phosphorylation in 293T cells treated with different doses of 8866 for 6 hours in the presence of DMSO or 5 μg/ml TM. Images shown are representative of 3 independent experiments. ( I ) Clonogenic growth of MCF10A MYC-ER cells transduced with GFP or XBP1s and treated with DMSO or 5 μM 8866 in the presence of different doses of 4-OHT. Changes in colony number were compared with vehicle-treated (ethanol and DMSO) MCF10A MYC-ER –GFP cells. In A and I , data are presented as mean ± SD of biological triplicates. * P

    Article Snippet: Nuclear extracts were incubated with 4 μg of anti-XBP1s antibody or anti-MYC antibody (catalog sc-42, Santa Cruz Biotechnology Inc.) overnight at 4°C.

    Techniques: Inhibition, Transduction, Expressing, Splicing Assay, Chromatin Immunoprecipitation, Fluorescence, Cleavage Assay, Incubation, SDS Page

    MYC is sufficient for activation of the IRE1/XBP1 pathway. ( A ) Schematic representation of the MCF10A MYC-ER system. In the presence of 4-OHT, MYC-ER fusion protein translocates to the nucleus and transactivates the MYC target genes. ( B ) Immunoblot and XBP1 splicing assay (RT-PCR) of MCF10A MYC-ER cells treated with different doses of 4-OHT for 24 hours. MYC-ER, XBP1s, and TBP were detected from nuclear extracts (NE) and IRE1 from whole cell lysates. TBP, actin, and GAPDH were used as loading control. ( C and D ) qRT-PCR analysis of the expression of IRE1 , XBP1s , XBP1 t , XBP1 s/t ( C ), and XBP1 target genes ( D ) in MCF10A MYC-ER cells treated with different doses of 4-OHT for 24 hours. ( E and F ) The tissue microarray containing specimens from 60 breast cancer patients was subjected to IHC for MYC and IRE1 (DAB staining, brown). ( E ) Representative photographs are shown indicating weak, moderate, and strong staining. Scale bars: 50 μm. ( F ) MYC H-score in tissue microarray samples with distinct IRE1 intensities. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. Tissue microarray was performed once, and all other data are representative of 3 independent experiments. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

    doi: 10.1172/JCI95873

    Figure Lengend Snippet: MYC is sufficient for activation of the IRE1/XBP1 pathway. ( A ) Schematic representation of the MCF10A MYC-ER system. In the presence of 4-OHT, MYC-ER fusion protein translocates to the nucleus and transactivates the MYC target genes. ( B ) Immunoblot and XBP1 splicing assay (RT-PCR) of MCF10A MYC-ER cells treated with different doses of 4-OHT for 24 hours. MYC-ER, XBP1s, and TBP were detected from nuclear extracts (NE) and IRE1 from whole cell lysates. TBP, actin, and GAPDH were used as loading control. ( C and D ) qRT-PCR analysis of the expression of IRE1 , XBP1s , XBP1 t , XBP1 s/t ( C ), and XBP1 target genes ( D ) in MCF10A MYC-ER cells treated with different doses of 4-OHT for 24 hours. ( E and F ) The tissue microarray containing specimens from 60 breast cancer patients was subjected to IHC for MYC and IRE1 (DAB staining, brown). ( E ) Representative photographs are shown indicating weak, moderate, and strong staining. Scale bars: 50 μm. ( F ) MYC H-score in tissue microarray samples with distinct IRE1 intensities. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. Tissue microarray was performed once, and all other data are representative of 3 independent experiments. * P

    Article Snippet: Nuclear extracts were incubated with 4 μg of anti-XBP1s antibody or anti-MYC antibody (catalog sc-42, Santa Cruz Biotechnology Inc.) overnight at 4°C.

    Techniques: Activation Assay, Splicing Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Microarray, Immunohistochemistry, Staining

    MYC interacts with XBP1s and regulates XBP1s transcriptional activity. ( A – C ) ChIP assays of SUM159 cells ( A ) and MC1 PDX tumors ( B and C ) were performed using anti-MYC or anti-XBP1s antibodies to detect enriched gene-promoter fragments. IgG was used as mock control. Genomic region upstream of VEGFA lacking XBP1s- and MYC-binding sites was used as a negative control (Ctrl). Data are presented relative to input and shown as mean ± SD of technical triplicates. ( D ) ChIP-re-ChIP assay of SUM159 cells was performed using the anti-MYC antibody first (MYC ChIP). Eluents were subjected to a second ChIP assay using IgG (IgG reChIP) or anti-XBP1s antibody (XBP1s reChIP) to detect enriched gene-promoter fragments. ND, not detected by qPCR assay. Data are shown as mean ± SD of technical triplicates. ( E and F ) Flag-tagged MYC and HA-tagged XBP1s were coexpressed in 293T cells and coimmunoprecipitation was performed with anti-HA antibody ( E ) or anti-Flag antibody ( F ). The immunoblot was probed with anti-Flag and anti-HA antibodies. HA-GFP ( E ) or Flag-GFP ( F ) was used as control. ( G and H ) Nuclear extracts from TM-treated SUM159 cells were subjected to coimmunoprecipitation with anti-XBP1s antibody ( G ) or anti-MYC antibody ( H ). The immunoblot was probed with anti-XBP1s and anti-MYC antibodies. ( I – L ) UPRE or ERSE reporter was cotransfected with MYC or XBP1s or both expression plasmids into BT549 ( I and K ) or 293T ( J and L ) cells. Luciferase activity was measured 48 hours after transfection. In I – L , GFP expression plasmid was used as control. Data are presented relative to Renilla readings and shown as mean ± SD of biological triplicates. All results shown are representative of 3 independent experiments. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

    doi: 10.1172/JCI95873

    Figure Lengend Snippet: MYC interacts with XBP1s and regulates XBP1s transcriptional activity. ( A – C ) ChIP assays of SUM159 cells ( A ) and MC1 PDX tumors ( B and C ) were performed using anti-MYC or anti-XBP1s antibodies to detect enriched gene-promoter fragments. IgG was used as mock control. Genomic region upstream of VEGFA lacking XBP1s- and MYC-binding sites was used as a negative control (Ctrl). Data are presented relative to input and shown as mean ± SD of technical triplicates. ( D ) ChIP-re-ChIP assay of SUM159 cells was performed using the anti-MYC antibody first (MYC ChIP). Eluents were subjected to a second ChIP assay using IgG (IgG reChIP) or anti-XBP1s antibody (XBP1s reChIP) to detect enriched gene-promoter fragments. ND, not detected by qPCR assay. Data are shown as mean ± SD of technical triplicates. ( E and F ) Flag-tagged MYC and HA-tagged XBP1s were coexpressed in 293T cells and coimmunoprecipitation was performed with anti-HA antibody ( E ) or anti-Flag antibody ( F ). The immunoblot was probed with anti-Flag and anti-HA antibodies. HA-GFP ( E ) or Flag-GFP ( F ) was used as control. ( G and H ) Nuclear extracts from TM-treated SUM159 cells were subjected to coimmunoprecipitation with anti-XBP1s antibody ( G ) or anti-MYC antibody ( H ). The immunoblot was probed with anti-XBP1s and anti-MYC antibodies. ( I – L ) UPRE or ERSE reporter was cotransfected with MYC or XBP1s or both expression plasmids into BT549 ( I and K ) or 293T ( J and L ) cells. Luciferase activity was measured 48 hours after transfection. In I – L , GFP expression plasmid was used as control. Data are presented relative to Renilla readings and shown as mean ± SD of biological triplicates. All results shown are representative of 3 independent experiments. * P

    Article Snippet: Nuclear extracts were incubated with 4 μg of anti-XBP1s antibody or anti-MYC antibody (catalog sc-42, Santa Cruz Biotechnology Inc.) overnight at 4°C.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Expressing, Luciferase, Transfection, Plasmid Preparation

    MYC is necessary for activation of the IRE1/XBP1 pathway. ( A and B ) Immunoblot of MYC and IRE1 in SUM159 cells ( A ) or BT549 cells ( B ) infected with lentiviruses encoding control scramble shRNA ( shScr ) or 2 distinct MYC shRNAs ( shMYC-1 and shMYC-2 ). Actin and GAPDH were used as loading controls. ( C – F ) qRT-PCR analysis of IRE1 expression and XBP1 splicing in infected SUM159 cells ( C and E ) or BT549 cells ( D and F ). XBP1 s/t , the ratio of XBP1s to total XBP1t . The XBP1 s/t ratio was normalized to that of the scramble ( shScr ) control. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. All data are representative of 3 independent experiments. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

    doi: 10.1172/JCI95873

    Figure Lengend Snippet: MYC is necessary for activation of the IRE1/XBP1 pathway. ( A and B ) Immunoblot of MYC and IRE1 in SUM159 cells ( A ) or BT549 cells ( B ) infected with lentiviruses encoding control scramble shRNA ( shScr ) or 2 distinct MYC shRNAs ( shMYC-1 and shMYC-2 ). Actin and GAPDH were used as loading controls. ( C – F ) qRT-PCR analysis of IRE1 expression and XBP1 splicing in infected SUM159 cells ( C and E ) or BT549 cells ( D and F ). XBP1 s/t , the ratio of XBP1s to total XBP1t . The XBP1 s/t ratio was normalized to that of the scramble ( shScr ) control. Data in qRT-PCR analysis are presented relative to actin and shown as mean ± SD of technical triplicates. All data are representative of 3 independent experiments. * P

    Article Snippet: Nuclear extracts were incubated with 4 μg of anti-XBP1s antibody or anti-MYC antibody (catalog sc-42, Santa Cruz Biotechnology Inc.) overnight at 4°C.

    Techniques: Activation Assay, Infection, shRNA, Quantitative RT-PCR, Expressing

    JAK2 (V617F)-induced cell proliferation and transformation require the expression of c-Myc. VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or c-Myc. (A) Transduced VF/EpoR cells were cultured without Epo for 12 hr. Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody or anti-β-actin antibody. (B) ODC mRNA was analyzed by quantitative real-time PCR. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (C) Transduced VF/EpoR cells were cultured without Epo for 3 days. Viable cells were counted and shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Transduced VF/EpoR cells were cultured without Epo for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (E-I) Transduced VF/EpoR cells were s.c. injected into nude mice (1×10 7 cells/mice). (E) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (F, G) Sixteen days post-inoculation, three mice were sacrificed. Morphological changes of the spleen and liver are shown in the photograph. The weights of the tumor, liver, and spleen were measured and plotted on the graph. * and ** indicate significant differences p

    Journal: PLoS ONE

    Article Title: Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

    doi: 10.1371/journal.pone.0052844

    Figure Lengend Snippet: JAK2 (V617F)-induced cell proliferation and transformation require the expression of c-Myc. VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or c-Myc. (A) Transduced VF/EpoR cells were cultured without Epo for 12 hr. Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody or anti-β-actin antibody. (B) ODC mRNA was analyzed by quantitative real-time PCR. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (C) Transduced VF/EpoR cells were cultured without Epo for 3 days. Viable cells were counted and shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Transduced VF/EpoR cells were cultured without Epo for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (E-I) Transduced VF/EpoR cells were s.c. injected into nude mice (1×10 7 cells/mice). (E) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (F, G) Sixteen days post-inoculation, three mice were sacrificed. Morphological changes of the spleen and liver are shown in the photograph. The weights of the tumor, liver, and spleen were measured and plotted on the graph. * and ** indicate significant differences p

    Article Snippet: Anti-β-actin antibody, anti-c-Myc antibody (N262), anti-EpoR antibody and anti-ODC antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Transformation Assay, Expressing, Infection, shRNA, Luciferase, Cell Culture, Real-time Polymerase Chain Reaction, FACS, Injection, Mouse Assay

    Inhibition of GSK-3β inhibited downregulation of c-Myc and apoptosis. (A) Transduced Ba/F3 cells were treated with DMSO (0.1%) or the indicated concentrations of GSK-3β inhibitor in the absence of IL-3 for 18 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (B-E) Transduced Ba/F3 cells were treated with DMSO (0.1%) or GSK-3β inhibitor (0.1 µM) for 18 hr. (B) Whole cell lysates were immunoblotted (IB) with anti-phospho-c-Myc antibody (Thr58), anti-c-Myc antibody or anti-β-actin antibody. (C) The expression amounts of c-Myc and its mutants were normalized with the protein amount of β-actin, and the quantified ratios of c-Myc and its mutants (T58A, In373) are shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Total RNA was prepared and mRNA of ectopic c-Myc and its mutants (T58A, In373) was detected by quantitative real time-PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (E) DNA was isolated from cells and subjected to agarose gel electrophoresis. (F, G) Ba/F3 cell lines were infected with empty virus (−), retrovirus encoding JAK2 mutant c-HA (V617F) and retroviruses encoding wild-type EpoR c-Flag (WT) or EpoR mutant c-Flag (Y479F). Transduced Ba/F3 cells were incubated without IL-3 for 12 hr. (F) Whole cell lysates were immunoblotted (IB) with anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-GSK-3β antibody (S9), anti-GSK-3β antibody, anti-c-Myc antibody, anti-Bcl-XL antibody, anti-HA antibody, anti-Flag antibody or anti-β-actin antibody. (G) c-Myc mRNA and Bcl-XL mRNA were analyzed by quantitative real-time PCR. GAPDH mRNA was evaluated as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments.

    Journal: PLoS ONE

    Article Title: Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

    doi: 10.1371/journal.pone.0052844

    Figure Lengend Snippet: Inhibition of GSK-3β inhibited downregulation of c-Myc and apoptosis. (A) Transduced Ba/F3 cells were treated with DMSO (0.1%) or the indicated concentrations of GSK-3β inhibitor in the absence of IL-3 for 18 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (B-E) Transduced Ba/F3 cells were treated with DMSO (0.1%) or GSK-3β inhibitor (0.1 µM) for 18 hr. (B) Whole cell lysates were immunoblotted (IB) with anti-phospho-c-Myc antibody (Thr58), anti-c-Myc antibody or anti-β-actin antibody. (C) The expression amounts of c-Myc and its mutants were normalized with the protein amount of β-actin, and the quantified ratios of c-Myc and its mutants (T58A, In373) are shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Total RNA was prepared and mRNA of ectopic c-Myc and its mutants (T58A, In373) was detected by quantitative real time-PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (E) DNA was isolated from cells and subjected to agarose gel electrophoresis. (F, G) Ba/F3 cell lines were infected with empty virus (−), retrovirus encoding JAK2 mutant c-HA (V617F) and retroviruses encoding wild-type EpoR c-Flag (WT) or EpoR mutant c-Flag (Y479F). Transduced Ba/F3 cells were incubated without IL-3 for 12 hr. (F) Whole cell lysates were immunoblotted (IB) with anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-GSK-3β antibody (S9), anti-GSK-3β antibody, anti-c-Myc antibody, anti-Bcl-XL antibody, anti-HA antibody, anti-Flag antibody or anti-β-actin antibody. (G) c-Myc mRNA and Bcl-XL mRNA were analyzed by quantitative real-time PCR. GAPDH mRNA was evaluated as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments.

    Article Snippet: Anti-β-actin antibody, anti-c-Myc antibody (N262), anti-EpoR antibody and anti-ODC antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Infection, Mutagenesis, Incubation

    JAK2 (V617F) induced expression of c-Myc through STAT5 activation. (A–E) Ba/F3 cell lines were infected with empty virus (−), retroviruses encoding wild-type JAK2 c-HA (WT), JAK2 mutant c-HA (V617F) and EpoR c-Flag as indicated in each figure. (A) WT/EpoR cells and VF/EpoR cells were cultured without Epo for 12 hr, and then total RNAs were prepared from each cell line. The enhancement of gene expression induced by JAK2 (V617F) was determined by DNA array. The ratio of the altered gene expressions in VF/EpoR cells was calculated by dividing the amount of each gene in VF/EpoR cells by their amount in WT/EpoR cells. (B, C) A series of Ba/F3 cells expressing the described genes was cultured untreated or stimulated with Epo (5 U/mL) for 12 hr. (D, E) A series of Ba/F3 cells expressing the indicated genes were cultured in the absence of Epo, and treated with DMSO (0.1%) or AG490 (30 µM) for 12 hr. (F) HEL cells were treated with DMSO (0.1%) or AG490 (10, 20, 30 µM) for 24 hr. (G, H) VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or STAT5. (I, J) Ba/F3 cells were infected with empty virus (−), retrovirus encoding wild-type STAT5 (WT) or the constitutively active mutant of STAT5 (1*6). The cells were cultured without Epo for 12 hr. (B, E, H, J) The mRNA expression of c-Myc and ODC was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three experiments. (C, D, F, G, I) Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody, anti-HA antibody, anti-EpoR antibody, anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-STAT5 antibody (Y694), anti-STAT5 antibody or anti-β-actin antibody.

    Journal: PLoS ONE

    Article Title: Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

    doi: 10.1371/journal.pone.0052844

    Figure Lengend Snippet: JAK2 (V617F) induced expression of c-Myc through STAT5 activation. (A–E) Ba/F3 cell lines were infected with empty virus (−), retroviruses encoding wild-type JAK2 c-HA (WT), JAK2 mutant c-HA (V617F) and EpoR c-Flag as indicated in each figure. (A) WT/EpoR cells and VF/EpoR cells were cultured without Epo for 12 hr, and then total RNAs were prepared from each cell line. The enhancement of gene expression induced by JAK2 (V617F) was determined by DNA array. The ratio of the altered gene expressions in VF/EpoR cells was calculated by dividing the amount of each gene in VF/EpoR cells by their amount in WT/EpoR cells. (B, C) A series of Ba/F3 cells expressing the described genes was cultured untreated or stimulated with Epo (5 U/mL) for 12 hr. (D, E) A series of Ba/F3 cells expressing the indicated genes were cultured in the absence of Epo, and treated with DMSO (0.1%) or AG490 (30 µM) for 12 hr. (F) HEL cells were treated with DMSO (0.1%) or AG490 (10, 20, 30 µM) for 24 hr. (G, H) VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or STAT5. (I, J) Ba/F3 cells were infected with empty virus (−), retrovirus encoding wild-type STAT5 (WT) or the constitutively active mutant of STAT5 (1*6). The cells were cultured without Epo for 12 hr. (B, E, H, J) The mRNA expression of c-Myc and ODC was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three experiments. (C, D, F, G, I) Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody, anti-HA antibody, anti-EpoR antibody, anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-STAT5 antibody (Y694), anti-STAT5 antibody or anti-β-actin antibody.

    Article Snippet: Anti-β-actin antibody, anti-c-Myc antibody (N262), anti-EpoR antibody and anti-ODC antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Expressing, Activation Assay, Infection, Mutagenesis, Cell Culture, DNA Array, shRNA, Luciferase, Real-time Polymerase Chain Reaction

    c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells. (A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.

    Journal: PLoS ONE

    Article Title: Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant

    doi: 10.1371/journal.pone.0052844

    Figure Lengend Snippet: c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells. (A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.

    Article Snippet: Anti-β-actin antibody, anti-c-Myc antibody (N262), anti-EpoR antibody and anti-ODC antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

    Techniques: Mutagenesis, Infection, Incubation, Cell Culture, Real-time Polymerase Chain Reaction, Expressing, FACS, Isolation, Agarose Gel Electrophoresis

    The N terminus of Rsp3 contains signals for processing and secretion. a Schematic representation of constructs used to analyze N-terminal processing of Rsp3. In all constructs, expression was driven from the constitutive otef promoter and all contain a C-terminal HA-tag. Amino acid sequences from 24 to 70 downstream of the signal peptide (SP) are shown. The position of Myc-tag insertion is depicted in light blue. Amino acids DGGA identified by N-terminal sequencing as N terminus of secreted Rsp3 are indicated in red. The arrow indicates the cleavage site. Amino acids subjected to alanine substitution are indicated in green. b Western blot analysis of Rsp3 secretion. SG200Δrsp3 strains expressing the indicated proteins were grown in CM liquid medium to an OD 600 of 0.6. Proteins from cell pellets and from supernatants (collected after TCA precipitation) were prepared subjected to western blot. The western blots were developed with either anti-HA or anti c-Myc antibodies as indicated. Detection of tubulin via an anti-tubulin antibody served as internal control for a cytosolic protein. c Secretion of Rsp3 variants carrying amino acid substitutions or deletions in the N-terminal domain. SG200Δrsp3 strains expressing the indicated proteins were analyzed by western blot after fractionation in supernatant and cell pellet as in b

    Journal: Nature Communications

    Article Title: The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins

    doi: 10.1038/s41467-018-04149-0

    Figure Lengend Snippet: The N terminus of Rsp3 contains signals for processing and secretion. a Schematic representation of constructs used to analyze N-terminal processing of Rsp3. In all constructs, expression was driven from the constitutive otef promoter and all contain a C-terminal HA-tag. Amino acid sequences from 24 to 70 downstream of the signal peptide (SP) are shown. The position of Myc-tag insertion is depicted in light blue. Amino acids DGGA identified by N-terminal sequencing as N terminus of secreted Rsp3 are indicated in red. The arrow indicates the cleavage site. Amino acids subjected to alanine substitution are indicated in green. b Western blot analysis of Rsp3 secretion. SG200Δrsp3 strains expressing the indicated proteins were grown in CM liquid medium to an OD 600 of 0.6. Proteins from cell pellets and from supernatants (collected after TCA precipitation) were prepared subjected to western blot. The western blots were developed with either anti-HA or anti c-Myc antibodies as indicated. Detection of tubulin via an anti-tubulin antibody served as internal control for a cytosolic protein. c Secretion of Rsp3 variants carrying amino acid substitutions or deletions in the N-terminal domain. SG200Δrsp3 strains expressing the indicated proteins were analyzed by western blot after fractionation in supernatant and cell pellet as in b

    Article Snippet: Proteins were detected in Western blot analyses using mouse monoclonal anti-HA (1:10,000 dilution, Sigma Cat#H9658), anti c-Myc (1:10,000 dilution, Sigma Cat#M4439), anti-tubulin (1:2000 dilution, Merck Cat#CP06), or rabbit anti-mCherry (1:3000 dilution, BioVision Cat#5993) antibodies, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:10,000 dilution, Cell Signaling) as secondary antibody.

    Techniques: Construct, Expressing, Sequencing, Western Blot, TCA Precipitation, Fractionation

    BAG2 forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and MYC-BAG2 were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection

    Journal: The Journal of Biological Chemistry

    Article Title: BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

    doi: 10.1074/jbc.M115.677815

    Figure Lengend Snippet: BAG2 forms a complex with PINK1. A , BAG2 interacts with PINK1 upon expression of PINK1 and BAG2 in HEK293T cells by co-immunoprecipitation. The plasmids PINK1-V5 and MYC-BAG2 were co-transfected into HEK293T cells. For IP of MYC-BAG2 ( left panel ), transfection

    Article Snippet: The following antibodies were used: rabbit anti-BAG2 (Abcam), rabbit anti-PINK1(494) (Novus), mouse anti-FLAG (Sigma), mouse anti-MYC and rabbit anti-RAF1 (Santa Cruz Biotechnology), mouse anti-V5 and anti-complex I (Invitrogen), mouse anti-ACTIN (Sigma), rabbit anti-TOM20 (Santa Cruz Biotechnology), mouse anti-UBIQUITIN (Abcam), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibody (eBiosciences) for IP Western blots and anti-mouse and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore) for regular Western blots.

    Techniques: Expressing, Immunoprecipitation, Transfection

    BAG2 regulates PARKIN translocation via PINK1. A and B , BAG2 expression regulates PARKIN translocation in HEK293T cells. HEK293T cells were cotransfected with empty vector and GFP , empty vector and GFP-PARKIN , MYC-BAG2 and GFP , or MYC-BAG2 and GFP-PARKIN

    Journal: The Journal of Biological Chemistry

    Article Title: BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

    doi: 10.1074/jbc.M115.677815

    Figure Lengend Snippet: BAG2 regulates PARKIN translocation via PINK1. A and B , BAG2 expression regulates PARKIN translocation in HEK293T cells. HEK293T cells were cotransfected with empty vector and GFP , empty vector and GFP-PARKIN , MYC-BAG2 and GFP , or MYC-BAG2 and GFP-PARKIN

    Article Snippet: The following antibodies were used: rabbit anti-BAG2 (Abcam), rabbit anti-PINK1(494) (Novus), mouse anti-FLAG (Sigma), mouse anti-MYC and rabbit anti-RAF1 (Santa Cruz Biotechnology), mouse anti-V5 and anti-complex I (Invitrogen), mouse anti-ACTIN (Sigma), rabbit anti-TOM20 (Santa Cruz Biotechnology), mouse anti-UBIQUITIN (Abcam), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibody (eBiosciences) for IP Western blots and anti-mouse and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore) for regular Western blots.

    Techniques: Translocation Assay, Expressing, Plasmid Preparation

    BAG2 regulates PINK1 stability. A , PINK1-FLAG and MYC-BAG2 were coexpressed in HEK293T cells. Expression of PINK1-FLAG or MYC-BAG2 was observed from total cell lysates ( left panel ). PINK1 was expressed in BAG2 knockdown HEK293T cells. Overexpressed PINK1-FLAG

    Journal: The Journal of Biological Chemistry

    Article Title: BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

    doi: 10.1074/jbc.M115.677815

    Figure Lengend Snippet: BAG2 regulates PINK1 stability. A , PINK1-FLAG and MYC-BAG2 were coexpressed in HEK293T cells. Expression of PINK1-FLAG or MYC-BAG2 was observed from total cell lysates ( left panel ). PINK1 was expressed in BAG2 knockdown HEK293T cells. Overexpressed PINK1-FLAG

    Article Snippet: The following antibodies were used: rabbit anti-BAG2 (Abcam), rabbit anti-PINK1(494) (Novus), mouse anti-FLAG (Sigma), mouse anti-MYC and rabbit anti-RAF1 (Santa Cruz Biotechnology), mouse anti-V5 and anti-complex I (Invitrogen), mouse anti-ACTIN (Sigma), rabbit anti-TOM20 (Santa Cruz Biotechnology), mouse anti-UBIQUITIN (Abcam), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibody (eBiosciences) for IP Western blots and anti-mouse and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore) for regular Western blots.

    Techniques: Expressing

    BAG2 protects neurons through PINK1 and PARKIN against MPP + challenge. A , expression or Knockdown of BAG2 in cortical neurons modulates neuronal viability. A plasmid expressing GFP or MYC-BAG2 was transfected into primary cortical neurons. Top panel ,

    Journal: The Journal of Biological Chemistry

    Article Title: BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

    doi: 10.1074/jbc.M115.677815

    Figure Lengend Snippet: BAG2 protects neurons through PINK1 and PARKIN against MPP + challenge. A , expression or Knockdown of BAG2 in cortical neurons modulates neuronal viability. A plasmid expressing GFP or MYC-BAG2 was transfected into primary cortical neurons. Top panel ,

    Article Snippet: The following antibodies were used: rabbit anti-BAG2 (Abcam), rabbit anti-PINK1(494) (Novus), mouse anti-FLAG (Sigma), mouse anti-MYC and rabbit anti-RAF1 (Santa Cruz Biotechnology), mouse anti-V5 and anti-complex I (Invitrogen), mouse anti-ACTIN (Sigma), rabbit anti-TOM20 (Santa Cruz Biotechnology), mouse anti-UBIQUITIN (Abcam), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibody (eBiosciences) for IP Western blots and anti-mouse and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore) for regular Western blots.

    Techniques: Expressing, Plasmid Preparation, Transfection

    BAG2 regulates PINK1 degradation in HEK293T cells. A , ubiquitination of overexpressed PINK1 is inhibited by BAG2. PINK1-FLAG or PINK1 ( K219M )- FLAG along with MYC-BAG2 was coexpressed in HEK293T cells. Transfection of PINK1-FLAG and an empty vector was

    Journal: The Journal of Biological Chemistry

    Article Title: BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

    doi: 10.1074/jbc.M115.677815

    Figure Lengend Snippet: BAG2 regulates PINK1 degradation in HEK293T cells. A , ubiquitination of overexpressed PINK1 is inhibited by BAG2. PINK1-FLAG or PINK1 ( K219M )- FLAG along with MYC-BAG2 was coexpressed in HEK293T cells. Transfection of PINK1-FLAG and an empty vector was

    Article Snippet: The following antibodies were used: rabbit anti-BAG2 (Abcam), rabbit anti-PINK1(494) (Novus), mouse anti-FLAG (Sigma), mouse anti-MYC and rabbit anti-RAF1 (Santa Cruz Biotechnology), mouse anti-V5 and anti-complex I (Invitrogen), mouse anti-ACTIN (Sigma), rabbit anti-TOM20 (Santa Cruz Biotechnology), mouse anti-UBIQUITIN (Abcam), and anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibody (eBiosciences) for IP Western blots and anti-mouse and anti-rabbit horse radish peroxidase-conjugated secondary antibodies (Millipore) for regular Western blots.

    Techniques: Transfection, Plasmid Preparation

    Slit2 expression did not affect the expression of N-cadherin and β-catenin in glioma cells. (A) Immunoprecipitation (IP) and immunoblot (IB) analyses. Cell lysates of parental (P) and Slit2-expressing cell clones of SNB19 and U373MG cells were subjected to IP using an anti-N-cadherin antibody followed by IB using anti-N-cadherin and anti-β-catenin antibodies. β-Catenin was effectively precipitated with N-cadherin in both parental and Slit2-expressing cells. (B) IB analyses. Human embryonic kidney 293T and human glioma SNB19 and U373MG parent cells (P) and their Slit2-expressing cell clones were analyzed for expression of Slit2, N-cadherin, and β-catenin by IB using anti-c-Myc, anti-N-cadherin, and anti-β-catenin antibodies. The molecular weights of proteins are, for Slit2 with c-Myc tags, ~250 kDa; N-cadherin, 136 kDa; and β-catenin, 92 kDa. β-Actin was used as a loading control. Slit2 expression significantly decreased expression of N-cadherin and β-catenin in 293T cells but did not affect the expression of these proteins in SNB19 and U373MG glioma cells. For A and B, results represent three independent experiments with similar results.

    Journal: Neuro-Oncology

    Article Title: Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity

    doi: 10.1215/15228517-2008-017

    Figure Lengend Snippet: Slit2 expression did not affect the expression of N-cadherin and β-catenin in glioma cells. (A) Immunoprecipitation (IP) and immunoblot (IB) analyses. Cell lysates of parental (P) and Slit2-expressing cell clones of SNB19 and U373MG cells were subjected to IP using an anti-N-cadherin antibody followed by IB using anti-N-cadherin and anti-β-catenin antibodies. β-Catenin was effectively precipitated with N-cadherin in both parental and Slit2-expressing cells. (B) IB analyses. Human embryonic kidney 293T and human glioma SNB19 and U373MG parent cells (P) and their Slit2-expressing cell clones were analyzed for expression of Slit2, N-cadherin, and β-catenin by IB using anti-c-Myc, anti-N-cadherin, and anti-β-catenin antibodies. The molecular weights of proteins are, for Slit2 with c-Myc tags, ~250 kDa; N-cadherin, 136 kDa; and β-catenin, 92 kDa. β-Actin was used as a loading control. Slit2 expression significantly decreased expression of N-cadherin and β-catenin in 293T cells but did not affect the expression of these proteins in SNB19 and U373MG glioma cells. For A and B, results represent three independent experiments with similar results.

    Article Snippet: The following antibodies were used in our studies: goat anti-β-actin (I-19, SC-1616) and mouse anti-c-Myc (9E10, SC-40 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-β-catenin (610153; BD Biosciences, San Diego, CA, USA); anti-N-cadherin (3B9, 33-3900; Invitrogen Life Science Zymed, Carlsbad, CA, USA); antiphospho-tyrosine (4G10, 05-321, Upstate Biotechnology, Lake Placid, NY, USA); and mouse anti-N-Cadherin (GC-4, C3865; Sigma Chemicals, St. Louis, MO, USA).

    Techniques: Expressing, Immunoprecipitation, Clone Assay

    Slit2 specifically inhibits Cdc42 activity and glioma cell motility in vitro. (A and B) Slit2 expression (A) or treatment with recombinant Slit2 (B) attenuated GTPase activity of Cdc42 but not the expression of Cdc42 proteins in glioma cells. (A) Slit2 expression attenuated Cdc42 activity in glioma cells. Total cell lysates of parental (P) SNB19 and U373MG cells and various Slit2-expressing cell clones were analyzed for guanosine-5′-triphosphate (GTP) loading of Cdc42 using a Cdc42 activity assessment assay kit. (B) Recombinant Slit2 specifically inhibits Cdc42 activity. Parental cells of SNB19 and U373MG were treated with conditioned media (CM) collected from their parental cells (P) or Slit2-expressing cell clones (S). In parallel, CM of Slit2-expressing cell clones were incubated with 20 μg/ml of an anti-c-Myc antibody overnight at 4°C followed by immunodepletion of Slit2 from the CM prior to the assays (S/Ab). The assays were performed using CM of SNB19/Slit2 clone 3 and 4 cells and U373MG/Slit2 clone 7 and 56 cells with similar results. In both A and B, the membranes were probed with anti-Cdc42 and anti-β-actin antibodies as loading controls. (C) In vitro cell migration. Treatment of parental SNB19 and U373MG cells with Slit2-containing CM reduces ability to migrate through the gelatin-coated membranes, whereas removal of Slit2 from CM by immunodepletion prior to the assays prevents Slit2 inhibition of cell migration in vitro. Error bars in C represent SD *p

    Journal: Neuro-Oncology

    Article Title: Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity

    doi: 10.1215/15228517-2008-017

    Figure Lengend Snippet: Slit2 specifically inhibits Cdc42 activity and glioma cell motility in vitro. (A and B) Slit2 expression (A) or treatment with recombinant Slit2 (B) attenuated GTPase activity of Cdc42 but not the expression of Cdc42 proteins in glioma cells. (A) Slit2 expression attenuated Cdc42 activity in glioma cells. Total cell lysates of parental (P) SNB19 and U373MG cells and various Slit2-expressing cell clones were analyzed for guanosine-5′-triphosphate (GTP) loading of Cdc42 using a Cdc42 activity assessment assay kit. (B) Recombinant Slit2 specifically inhibits Cdc42 activity. Parental cells of SNB19 and U373MG were treated with conditioned media (CM) collected from their parental cells (P) or Slit2-expressing cell clones (S). In parallel, CM of Slit2-expressing cell clones were incubated with 20 μg/ml of an anti-c-Myc antibody overnight at 4°C followed by immunodepletion of Slit2 from the CM prior to the assays (S/Ab). The assays were performed using CM of SNB19/Slit2 clone 3 and 4 cells and U373MG/Slit2 clone 7 and 56 cells with similar results. In both A and B, the membranes were probed with anti-Cdc42 and anti-β-actin antibodies as loading controls. (C) In vitro cell migration. Treatment of parental SNB19 and U373MG cells with Slit2-containing CM reduces ability to migrate through the gelatin-coated membranes, whereas removal of Slit2 from CM by immunodepletion prior to the assays prevents Slit2 inhibition of cell migration in vitro. Error bars in C represent SD *p

    Article Snippet: The following antibodies were used in our studies: goat anti-β-actin (I-19, SC-1616) and mouse anti-c-Myc (9E10, SC-40 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-β-catenin (610153; BD Biosciences, San Diego, CA, USA); anti-N-cadherin (3B9, 33-3900; Invitrogen Life Science Zymed, Carlsbad, CA, USA); antiphospho-tyrosine (4G10, 05-321, Upstate Biotechnology, Lake Placid, NY, USA); and mouse anti-N-Cadherin (GC-4, C3865; Sigma Chemicals, St. Louis, MO, USA).

    Techniques: Activity Assay, In Vitro, Expressing, Recombinant, Clone Assay, Incubation, Migration, Inhibition

    Expression of Slit2 and Robo1 in primary glioma specimens and glioma cell lines. (A) Slit2 and Robo1 are expressed at lower levels in primary human glioma specimens and invasive SNB19 and U373MG glioma cells compared with normal human brain tissue (NB) and normal human astrocytes (NHA). Synthesized first-strand cDNA from total RNA of NB, primary glioma specimens (J4, J5, glioblastoma multiforme, WHO grade IV; J15 and J16, anaplastic oligodendroglioma, grade III), NHA, and SNB19 and U373MG glioma cells was used for PCR reactions. The length of synthesized cDNA fragments is 514 bp for Robo1 and 387 bp for Slit2, respectively. β-Actin was used as internal control. (B) Ectopic expression of Slit2 in SNB19 and U373MG glioma cells. Cell lysates (top two panels) or conditioned media (CM, bottom panel) from SNB19, SNB19 Slit2-expressing, U373MG, or U373MG Slit2-expressing cells were analyzed by immunoblot using an anti-c-Myc antibody. Lanes 2, 3, and 4 are SNB19 Slit2-expressing cell clones; lanes 7, 56, and 38 are U373MG Slit2-expressing cell clones. β-Actin was used as a loading control. Results shown in A and B are representative of three independent experiments.

    Journal: Neuro-Oncology

    Article Title: Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity

    doi: 10.1215/15228517-2008-017

    Figure Lengend Snippet: Expression of Slit2 and Robo1 in primary glioma specimens and glioma cell lines. (A) Slit2 and Robo1 are expressed at lower levels in primary human glioma specimens and invasive SNB19 and U373MG glioma cells compared with normal human brain tissue (NB) and normal human astrocytes (NHA). Synthesized first-strand cDNA from total RNA of NB, primary glioma specimens (J4, J5, glioblastoma multiforme, WHO grade IV; J15 and J16, anaplastic oligodendroglioma, grade III), NHA, and SNB19 and U373MG glioma cells was used for PCR reactions. The length of synthesized cDNA fragments is 514 bp for Robo1 and 387 bp for Slit2, respectively. β-Actin was used as internal control. (B) Ectopic expression of Slit2 in SNB19 and U373MG glioma cells. Cell lysates (top two panels) or conditioned media (CM, bottom panel) from SNB19, SNB19 Slit2-expressing, U373MG, or U373MG Slit2-expressing cells were analyzed by immunoblot using an anti-c-Myc antibody. Lanes 2, 3, and 4 are SNB19 Slit2-expressing cell clones; lanes 7, 56, and 38 are U373MG Slit2-expressing cell clones. β-Actin was used as a loading control. Results shown in A and B are representative of three independent experiments.

    Article Snippet: The following antibodies were used in our studies: goat anti-β-actin (I-19, SC-1616) and mouse anti-c-Myc (9E10, SC-40 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-β-catenin (610153; BD Biosciences, San Diego, CA, USA); anti-N-cadherin (3B9, 33-3900; Invitrogen Life Science Zymed, Carlsbad, CA, USA); antiphospho-tyrosine (4G10, 05-321, Upstate Biotechnology, Lake Placid, NY, USA); and mouse anti-N-Cadherin (GC-4, C3865; Sigma Chemicals, St. Louis, MO, USA).

    Techniques: Expressing, Synthesized, Polymerase Chain Reaction, Clone Assay

    Influence of β-TrCP overexpression or suppression on steady-state level and polyubiquitination of CREB-H-ΔTC. ( A ) Immunoblotting of cell lysates derived from HEK293T cells expressing β-TrCP (FLAG-β-T) or β-TrCP-ΔFbox (FLAG-ΔF), ubiquitin (Ub) and CREB-H-ΔTC (ΔTC). ( B ) Immunoblotting of cell lysates derived from HEK293T cells transfected with two independent siRNAs targeting β-TrCP (siβ-T#1 and siβ-T#2). siGFP served as a control. ( C ) Analysis of protein stability by cycloheximide chase assay. HEK293T cells cotransfected with siβ-T#1 and CREB-H-ΔTC plasmid were treated with 200 μM cycloheximide (CHX) for 3, 5 and 8 hours before harvest. Protein samples were analyzed by SDS-PAGE and probed with the indicated antibodies. ( D ) Polyubiquitination analysis. Lysates from HEK293T cells expressing the indicated proteins were immunoprecipitated with anti-V5 and the precipitates were analyzed by immunoblotting with anti-MYC, anti-FLAG and anti-V5. The inputs (10%) were probed with anti-V5 and anti-GAPDH.

    Journal: Scientific Reports

    Article Title: β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    doi: 10.1038/srep23938

    Figure Lengend Snippet: Influence of β-TrCP overexpression or suppression on steady-state level and polyubiquitination of CREB-H-ΔTC. ( A ) Immunoblotting of cell lysates derived from HEK293T cells expressing β-TrCP (FLAG-β-T) or β-TrCP-ΔFbox (FLAG-ΔF), ubiquitin (Ub) and CREB-H-ΔTC (ΔTC). ( B ) Immunoblotting of cell lysates derived from HEK293T cells transfected with two independent siRNAs targeting β-TrCP (siβ-T#1 and siβ-T#2). siGFP served as a control. ( C ) Analysis of protein stability by cycloheximide chase assay. HEK293T cells cotransfected with siβ-T#1 and CREB-H-ΔTC plasmid were treated with 200 μM cycloheximide (CHX) for 3, 5 and 8 hours before harvest. Protein samples were analyzed by SDS-PAGE and probed with the indicated antibodies. ( D ) Polyubiquitination analysis. Lysates from HEK293T cells expressing the indicated proteins were immunoprecipitated with anti-V5 and the precipitates were analyzed by immunoblotting with anti-MYC, anti-FLAG and anti-V5. The inputs (10%) were probed with anti-V5 and anti-GAPDH.

    Article Snippet: Primary antibodies used in this study included mouse anti-V5 (Invitrogen), rabbit anti-FLAG (F7425; Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), rabbit anti-MYC (A-14; Santa Cruz), rabbit anti-glyceraldehyde-3-phosphatase dehydrogenase (anti-GAPDH: H12; Santa Cruz), rabbit anti-β-TrCP (D13F10; Cell Signaling) and rabbit anti-β-actin (Sigma-Aldrich).

    Techniques: Over Expression, Derivative Assay, Expressing, Transfection, Plasmid Preparation, SDS Page, Immunoprecipitation

    Interaction between CREB-H-ΔTC and SCF β-TrCP . ( A ) Sequence alignment of CREB-H homologs from different species. The predicted β-TrCP-recognizing motif (DSGϕS) and the adjacent highly conserved serine-threonine residues are framed and highlighted. Identical residues are denoted by asterisks (*), whereas colons (:) and periods (.) indicate strong and weak similarity, respectively. ϕ: hydrophobic residue. x: any residue. Hs: Homo sapiens (human). Mm: Mus musculus (house mouse). Rn: Rattus norvegicus (Norway rat). Bt: Bos taurus (bovine). Dr: Danio rerio (zebrafish). Gg: Gallus gallus (chicken). ( B ) Sequence alignment of CREB-H with known β-TrCP substrates (upper) and other CREB3 subfamily proteins (lower). The β-TrCP recognizing motif is indicated. EPOR: erythropoietin receptor. RV-NSP1: rotavirus nonstructural protein NSP1. ( C,D ) Interaction between CREB-H-ΔTC and β-TrCP. Reciprocal co-immunoprecipitation was performed with V5-CREB-H-ΔTC (V5-ΔTC) and FLAG-β-TrCP (FLAG-β-T) or FLAG-CREB-H-ΔTC (FLAG-ΔTC) and V5-β-TrCP (V5-β-T) expressed in HEK293T cells. Cell lysates were immunoprecipitated with anti-V5 and the precipitates were immunoblotted with anti-FLAG and anti-V5. The input cell lysates (10%) were also probed with anti-FLAG and anti-V5. ( E,F ) Interaction of CREB-H-ΔTC with CUL1 and RBX1. Lysates from transfected HEK293T cells were immunoprecipitated with anti-V5 and the precipitates were analyzed by immunoblotting with anti-V5 and anti-MYC. The input lysates (10%) were also detected with anti-V5 and anti-MYC. ( G ) Interaction of CREB-HΔTC with β-TrCP, CUL1 and RBX1. The anti-V5 immunoprecipitates were probed with anti-MYC, anti-FLAG and anti-V5.

    Journal: Scientific Reports

    Article Title: β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    doi: 10.1038/srep23938

    Figure Lengend Snippet: Interaction between CREB-H-ΔTC and SCF β-TrCP . ( A ) Sequence alignment of CREB-H homologs from different species. The predicted β-TrCP-recognizing motif (DSGϕS) and the adjacent highly conserved serine-threonine residues are framed and highlighted. Identical residues are denoted by asterisks (*), whereas colons (:) and periods (.) indicate strong and weak similarity, respectively. ϕ: hydrophobic residue. x: any residue. Hs: Homo sapiens (human). Mm: Mus musculus (house mouse). Rn: Rattus norvegicus (Norway rat). Bt: Bos taurus (bovine). Dr: Danio rerio (zebrafish). Gg: Gallus gallus (chicken). ( B ) Sequence alignment of CREB-H with known β-TrCP substrates (upper) and other CREB3 subfamily proteins (lower). The β-TrCP recognizing motif is indicated. EPOR: erythropoietin receptor. RV-NSP1: rotavirus nonstructural protein NSP1. ( C,D ) Interaction between CREB-H-ΔTC and β-TrCP. Reciprocal co-immunoprecipitation was performed with V5-CREB-H-ΔTC (V5-ΔTC) and FLAG-β-TrCP (FLAG-β-T) or FLAG-CREB-H-ΔTC (FLAG-ΔTC) and V5-β-TrCP (V5-β-T) expressed in HEK293T cells. Cell lysates were immunoprecipitated with anti-V5 and the precipitates were immunoblotted with anti-FLAG and anti-V5. The input cell lysates (10%) were also probed with anti-FLAG and anti-V5. ( E,F ) Interaction of CREB-H-ΔTC with CUL1 and RBX1. Lysates from transfected HEK293T cells were immunoprecipitated with anti-V5 and the precipitates were analyzed by immunoblotting with anti-V5 and anti-MYC. The input lysates (10%) were also detected with anti-V5 and anti-MYC. ( G ) Interaction of CREB-HΔTC with β-TrCP, CUL1 and RBX1. The anti-V5 immunoprecipitates were probed with anti-MYC, anti-FLAG and anti-V5.

    Article Snippet: Primary antibodies used in this study included mouse anti-V5 (Invitrogen), rabbit anti-FLAG (F7425; Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), rabbit anti-MYC (A-14; Santa Cruz), rabbit anti-glyceraldehyde-3-phosphatase dehydrogenase (anti-GAPDH: H12; Santa Cruz), rabbit anti-β-TrCP (D13F10; Cell Signaling) and rabbit anti-β-actin (Sigma-Aldrich).

    Techniques: Sequencing, Immunoprecipitation, Transfection

    CREB-H-ΔTC-Δ81-90 was resistant to β-TrCP-induced degradation and polyubiquitination. ( A ) Protein stability. HepG2 cells expressing the indicated proteins were treated with 200 μM cycloheximide (CHX) for 1, 3 and 5 hours before harvest. Protein samples were collected, analyzed by SDS-PAGE and immunoblotted with anti-FLAG, anti-V5 and anti-GAPDH. ( B ) Steady-state protein expression. CREB-H-ΔTC (V5-ΔTC) or CREB-H-ΔTC-Δ81-90 (V5-Δ81-90), β-TrCP (FLAG-β-T) and ubiquitin (Ub) constructs were transfected into HEK293T cells. Cell lysates were immunoblotted with anti-V5 and anti-FLAG. GAPDH was detected as an internal control. ( C,D ) Immunoprecipitation. CREB-H-ΔTC (V5-ΔTC) or CREB-H-ΔTC-Δ81-90 (V5-Δ81-90) construct was transfected into HEK293T cells or HEK293T cells expressing FLAG-β-TrCP (FLAG-β-T). Cell lysates were immunoprecipitated with anti-V5. The inputs (10%) and the immunoprecipitates were analyzed by SDS-PAGE and probed with anti-V5 and either anti-β-TrCP or anti-FLAG. ( E ) In vivo polyubiquitination. HEK293T cells were transfected with constructs expressing the indicated proteins. Cell lysates were immunoprecipitated with anti-V5 and the precipitates were probed with anti-MYC, anti-FLAG and anti-V5. The input samples (10%) were probed with anti-FLAG, anti-V5 and anti-GAPDH.

    Journal: Scientific Reports

    Article Title: β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    doi: 10.1038/srep23938

    Figure Lengend Snippet: CREB-H-ΔTC-Δ81-90 was resistant to β-TrCP-induced degradation and polyubiquitination. ( A ) Protein stability. HepG2 cells expressing the indicated proteins were treated with 200 μM cycloheximide (CHX) for 1, 3 and 5 hours before harvest. Protein samples were collected, analyzed by SDS-PAGE and immunoblotted with anti-FLAG, anti-V5 and anti-GAPDH. ( B ) Steady-state protein expression. CREB-H-ΔTC (V5-ΔTC) or CREB-H-ΔTC-Δ81-90 (V5-Δ81-90), β-TrCP (FLAG-β-T) and ubiquitin (Ub) constructs were transfected into HEK293T cells. Cell lysates were immunoblotted with anti-V5 and anti-FLAG. GAPDH was detected as an internal control. ( C,D ) Immunoprecipitation. CREB-H-ΔTC (V5-ΔTC) or CREB-H-ΔTC-Δ81-90 (V5-Δ81-90) construct was transfected into HEK293T cells or HEK293T cells expressing FLAG-β-TrCP (FLAG-β-T). Cell lysates were immunoprecipitated with anti-V5. The inputs (10%) and the immunoprecipitates were analyzed by SDS-PAGE and probed with anti-V5 and either anti-β-TrCP or anti-FLAG. ( E ) In vivo polyubiquitination. HEK293T cells were transfected with constructs expressing the indicated proteins. Cell lysates were immunoprecipitated with anti-V5 and the precipitates were probed with anti-MYC, anti-FLAG and anti-V5. The input samples (10%) were probed with anti-FLAG, anti-V5 and anti-GAPDH.

    Article Snippet: Primary antibodies used in this study included mouse anti-V5 (Invitrogen), rabbit anti-FLAG (F7425; Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), rabbit anti-MYC (A-14; Santa Cruz), rabbit anti-glyceraldehyde-3-phosphatase dehydrogenase (anti-GAPDH: H12; Santa Cruz), rabbit anti-β-TrCP (D13F10; Cell Signaling) and rabbit anti-β-actin (Sigma-Aldrich).

    Techniques: Expressing, SDS Page, Construct, Transfection, Immunoprecipitation, In Vivo

    Degradation of CREB-H-ΔTC by the ubiquitin-proteasome pathway. ( A ) HEK293T cells expressing V5-tagged CREB-H-ΔTC (V5-ΔTC) were treated with 10 μM MG132 for 0.5, 1, 2 and 6 hours before harvest. Protein samples were separated by SDS-PAGE and probed with anti-V5 and anti-β-actin. ( B,C ) MYC-ubiquitin (MYC-Ub) and the indicated mutants were individually cotransfected with CREB-H-ΔTC or CREB-H into HEK293T cells. Immunoprecipitation was performed with anti-V5. Precipitates and inputs (10%) were analyzed by immunoblotting with anti-MYC. GAPDH was detected as an internal control. Ub: ubiquitin. K0: lysine free ubiquitin. 48R: ubiquitin with replacement of lysine 48 by arginine. 63R: ubiquitin with replacement of lysine 63 by arginine.

    Journal: Scientific Reports

    Article Title: β-TrCP-mediated ubiquitination and degradation of liver-enriched transcription factor CREB-H

    doi: 10.1038/srep23938

    Figure Lengend Snippet: Degradation of CREB-H-ΔTC by the ubiquitin-proteasome pathway. ( A ) HEK293T cells expressing V5-tagged CREB-H-ΔTC (V5-ΔTC) were treated with 10 μM MG132 for 0.5, 1, 2 and 6 hours before harvest. Protein samples were separated by SDS-PAGE and probed with anti-V5 and anti-β-actin. ( B,C ) MYC-ubiquitin (MYC-Ub) and the indicated mutants were individually cotransfected with CREB-H-ΔTC or CREB-H into HEK293T cells. Immunoprecipitation was performed with anti-V5. Precipitates and inputs (10%) were analyzed by immunoblotting with anti-MYC. GAPDH was detected as an internal control. Ub: ubiquitin. K0: lysine free ubiquitin. 48R: ubiquitin with replacement of lysine 48 by arginine. 63R: ubiquitin with replacement of lysine 63 by arginine.

    Article Snippet: Primary antibodies used in this study included mouse anti-V5 (Invitrogen), rabbit anti-FLAG (F7425; Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), rabbit anti-MYC (A-14; Santa Cruz), rabbit anti-glyceraldehyde-3-phosphatase dehydrogenase (anti-GAPDH: H12; Santa Cruz), rabbit anti-β-TrCP (D13F10; Cell Signaling) and rabbit anti-β-actin (Sigma-Aldrich).

    Techniques: Expressing, SDS Page, Immunoprecipitation

    The interactions of mlc1 mutant proteins with Myo2p, Iqg1p, and Myo1p. (A) Myo2p-MYC was precipitated with anti-MYC-agarose beads from cell lysates of strains YJL176A ( MYO1:HA MYO2:MYC ) carrying pUG34 (vector), pUG34-MLC1 (Mlc1p), pUG34-MLC1-93 (mlc1-93p), or pUG34-MLC1-11 (mlc1-11p), respectively. Myo2p-MYC, Mlc1p-GFP, and its derivatives in the bound and in the input fractions were detected by Western-blot analysis with antibodies against MYC or GFP as indicated in A. (B) As in A except that strain YJL175A ( myo1 Δ IQG1:HA MYO2:MYC ) carrying the different plasmids and the anti-HA-agarose beads were used to do the pull down. (C) As in A except that anti-HA-agarose beads were used to do the pull down. (D) Summary of the interactions between Mlc1p derivatives and its binding partners. (E) Position of the mlc1-11 and mlc1-93 mutations on the three-dimensional structure of the IQ2 (from Myo2p)–Mlc1p complex ( Terrak et al., 2003 ).

    Journal: The Journal of Cell Biology

    Article Title: Identification and functional analysis of the essential and regulatory light chains of the only type II myosin Myo1p in Saccharomyces cerevisiae

    doi: 10.1083/jcb.200401040

    Figure Lengend Snippet: The interactions of mlc1 mutant proteins with Myo2p, Iqg1p, and Myo1p. (A) Myo2p-MYC was precipitated with anti-MYC-agarose beads from cell lysates of strains YJL176A ( MYO1:HA MYO2:MYC ) carrying pUG34 (vector), pUG34-MLC1 (Mlc1p), pUG34-MLC1-93 (mlc1-93p), or pUG34-MLC1-11 (mlc1-11p), respectively. Myo2p-MYC, Mlc1p-GFP, and its derivatives in the bound and in the input fractions were detected by Western-blot analysis with antibodies against MYC or GFP as indicated in A. (B) As in A except that strain YJL175A ( myo1 Δ IQG1:HA MYO2:MYC ) carrying the different plasmids and the anti-HA-agarose beads were used to do the pull down. (C) As in A except that anti-HA-agarose beads were used to do the pull down. (D) Summary of the interactions between Mlc1p derivatives and its binding partners. (E) Position of the mlc1-11 and mlc1-93 mutations on the three-dimensional structure of the IQ2 (from Myo2p)–Mlc1p complex ( Terrak et al., 2003 ).

    Article Snippet: The precipitated Mlc2p-Myc or Mlc1p-HA, the total Myo1p-GFP variants in supernatants, and the bound fractions of Myo1p-GFP variants to the anti-MYC or anti-HA beads were separated by SDS-PAGE and blotted with anti-MYC, anti-HA, and anti-GFP mAbs, respectively (Covance Research Products Inc.).

    Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Binding Assay

    Mlc2p binds to IQ2 of Myo1p exclusively. (A) Diagram of IQ mutants of Myo1p. Asterisk indicates R806A change in IQ1. (B) Mlc2p binds to Myo1p through IQ2. Strain YEF3175 ( a myo1 Δ ::Kan MLC2:MYC ) was transformed individually with plasmid pRS316NoNot (Vector), pRS316-MYO1 (untagged Myo1p), and pRS316-N-MYO1-GFP or its IQ mutant derivatives. Transformants were grown exponentially in SC-Ura media at 24°C and processed for coIP experiments. Mlc2p-MYC: Mlc2p-MYC in the immunoprecipitates brought down by anti-MYC–conjugated agarose beads from different cell lysates; Myo1p-GFP-input: Myo1p-GFP in different cell lysates before immunoprecipitation; Myo1p-GFP-bound: Myo1p-GFP in the immunoprecipitates from different cell lysates. White lines indicate that intervening lanes have been spliced out. (C) Mlc2p fails to localize to the bud neck in cells carrying myo1-IQ2 Δ. YEF2603 ( myo1 Δ /myo1 Δ MLC2:GFP/MLC2:GFP ) was transformed individually with plasmid pRS316-N-MYO1-R806A-GFP (R806A), pRS316-N-MYO1-IQ1Δ-GFP (IQ1Δ), pRS316-N-MYO1-IQ2Δ-GFP (IQ2Δ), or pRS316-N-MYO1-(IQ1Δ+IQ2Δ)-GFP (IQ1Δ+IQ2Δ). Transformants were grown exponentially in SC-Ura media, fixed with formaldehyde, and observed with DIC and fluorescence microscopy.

    Journal: The Journal of Cell Biology

    Article Title: Identification and functional analysis of the essential and regulatory light chains of the only type II myosin Myo1p in Saccharomyces cerevisiae

    doi: 10.1083/jcb.200401040

    Figure Lengend Snippet: Mlc2p binds to IQ2 of Myo1p exclusively. (A) Diagram of IQ mutants of Myo1p. Asterisk indicates R806A change in IQ1. (B) Mlc2p binds to Myo1p through IQ2. Strain YEF3175 ( a myo1 Δ ::Kan MLC2:MYC ) was transformed individually with plasmid pRS316NoNot (Vector), pRS316-MYO1 (untagged Myo1p), and pRS316-N-MYO1-GFP or its IQ mutant derivatives. Transformants were grown exponentially in SC-Ura media at 24°C and processed for coIP experiments. Mlc2p-MYC: Mlc2p-MYC in the immunoprecipitates brought down by anti-MYC–conjugated agarose beads from different cell lysates; Myo1p-GFP-input: Myo1p-GFP in different cell lysates before immunoprecipitation; Myo1p-GFP-bound: Myo1p-GFP in the immunoprecipitates from different cell lysates. White lines indicate that intervening lanes have been spliced out. (C) Mlc2p fails to localize to the bud neck in cells carrying myo1-IQ2 Δ. YEF2603 ( myo1 Δ /myo1 Δ MLC2:GFP/MLC2:GFP ) was transformed individually with plasmid pRS316-N-MYO1-R806A-GFP (R806A), pRS316-N-MYO1-IQ1Δ-GFP (IQ1Δ), pRS316-N-MYO1-IQ2Δ-GFP (IQ2Δ), or pRS316-N-MYO1-(IQ1Δ+IQ2Δ)-GFP (IQ1Δ+IQ2Δ). Transformants were grown exponentially in SC-Ura media, fixed with formaldehyde, and observed with DIC and fluorescence microscopy.

    Article Snippet: The precipitated Mlc2p-Myc or Mlc1p-HA, the total Myo1p-GFP variants in supernatants, and the bound fractions of Myo1p-GFP variants to the anti-MYC or anti-HA beads were separated by SDS-PAGE and blotted with anti-MYC, anti-HA, and anti-GFP mAbs, respectively (Covance Research Products Inc.).

    Techniques: Transformation Assay, Plasmid Preparation, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Fluorescence, Microscopy