hxk2 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Santa Cruz Biotechnology anti hxk2 antibody
    Interaction of <t>Hxk2</t> wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.
    Anti Hxk2 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hxk2 antibody/product/Santa Cruz Biotechnology
    Average 88 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    anti hxk2 antibody - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    92
    Novus Biologicals anti hexokinase hxk2 hrp
    Interaction of <t>Hxk2</t> wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.
    Anti Hexokinase Hxk2 Hrp, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hexokinase hxk2 hrp/product/Novus Biologicals
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    anti hexokinase hxk2 hrp - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    86
    Alpha Diagnostics anti hexokinase ii hxk2 hk2 antibody
    Interaction of <t>Hxk2</t> wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.
    Anti Hexokinase Ii Hxk2 Hk2 Antibody, supplied by Alpha Diagnostics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hexokinase ii hxk2 hk2 antibody/product/Alpha Diagnostics
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hexokinase ii hxk2 hk2 antibody - by Bioz Stars, 2020-09
    86/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc anti hxk2 rabbit mab
    Induction of Glut1 and <t>HXK2</t> in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p
    Anti Hxk2 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti hxk2 rabbit mab/product/Cell Signaling Technology Inc
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti hxk2 rabbit mab - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology goat polyclonal anti hxk2
    Induction of Glut1 and <t>HXK2</t> in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p
    Goat Polyclonal Anti Hxk2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat polyclonal anti hxk2/product/Santa Cruz Biotechnology
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    goat polyclonal anti hxk2 - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    84
    Cell Signaling Technology Inc rabbit anti hxk 2
    Induction of Glut1 and <t>HXK2</t> in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p
    Rabbit Anti Hxk 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti hxk 2/product/Cell Signaling Technology Inc
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti hxk 2 - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    84
    US Biological Life Sciences rabbit α hexokinase α hxk2 antibody
    Induction of Glut1 and <t>HXK2</t> in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p
    Rabbit α Hexokinase α Hxk2 Antibody, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit α hexokinase α hxk2 antibody/product/US Biological Life Sciences
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit α hexokinase α hxk2 antibody - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    Interaction of Hxk2 wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Interaction of Hxk2 wca and Hxk2 wrf with Mig1 ( A ) In vivo co-immunoprecipitation of HA–Mig1 with Hxk2 wca and Hxk2 wrf . The W303.1A, W303.1A wca and W303.1A wrf strains, transformed with plasmid pWS93/Mig1 which encoded an HA-tagged Mig1 protein, were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody (lanes 1–6) or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Mig1 was visualized on a Western blot with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody. ( B ) Mig1 phosphorylation in response to Hxk2 wca and Hxk2 wrf availability. Cells, from wild-type (WT), Hxk2 wca and Hxk2 wrf strains transformed with the HA–Mig1 construct (plasmid pWS93/Mig1), were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The Mig1 protein was detected from total cell extracts by SDS/12% PAGE followed by immunoblotting with an anti-HA antibody. The phosphorylated forms of Mig1 are indicated as HA-Mig1-P (phosphorylated) and HA-Mig1 (dephosphorylated). ( C ) GST pull-down assays of the interaction of Hxk2 wca and Hxk2 wrf proteins with Mig1. A GST–Mig1 fusion protein was purified on glutathione–Sepharose columns. Equal amounts of GST–Mig1 were incubated with cell extracts from a wild-type strain (W303.1A) and the mutant strains W303.1A wca and W303.1A wrf . The yeast strains were grown in YEPG medium until a D 600 of 0.6 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. For the control samples, GST protein was also incubated with the H-Glc and L-Glc cell extracts, but no signals were detected (lanes 5 and 6). ( D ) The level of Hxk2 present in the different extracts used in ( C ) was determined by Western blotting using an anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: In Vivo, Immunoprecipitation, Transformation Assay, Plasmid Preparation, Selection, Gas Chromatography, Polyacrylamide Gel Electrophoresis, Western Blot, Construct, Purification, Incubation, Mutagenesis

    In vivo binding of Hxk2 wca to the MIG1 element of the SUC2 promoter ( A ) Wild-type (WT) W303.1A, the mutant strains W303.1A wca and W303.1A wrf and the double-mutant strain DBY2052 (Δ hxk1 Δ hxk2 ) were grown to mid-exponential phase in YEPG medium and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. Cell extracts were prepared, immunoprecipitated with anti-Hxk2 antibodies and the DNA fragments were amplified by PCR using the combination of oligonucleotides indicated in the Materials and methods section. The amplified fragments were resolved by agarose gel electrophoresis. Migration of standard markers is indicated on the right. A ChIP assay representative of three independent experiments is shown. ( B ) Verification of expression of the wild-type and Hxk2 mutant proteins. The double-mutant strain DBY2052 (Δ hxk1 Δ hxk2GLK1 ) lacking the two fructose-phosphorylating enzymes was used as control. Yeasts were grown to mid-exponential phase in complex medium with galactose, and then shifted to high-glucose medium for 1 h. The cells were harvested and lysed in 1% SDS/PBS containing protease inhibitors. Aliquots of 20 μg of protein/lane were resolved by SDS/12% PAGE. Hxk2 detection was performed using an anti-Hxk2 antibody.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: In vivo binding of Hxk2 wca to the MIG1 element of the SUC2 promoter ( A ) Wild-type (WT) W303.1A, the mutant strains W303.1A wca and W303.1A wrf and the double-mutant strain DBY2052 (Δ hxk1 Δ hxk2 ) were grown to mid-exponential phase in YEPG medium and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. Cell extracts were prepared, immunoprecipitated with anti-Hxk2 antibodies and the DNA fragments were amplified by PCR using the combination of oligonucleotides indicated in the Materials and methods section. The amplified fragments were resolved by agarose gel electrophoresis. Migration of standard markers is indicated on the right. A ChIP assay representative of three independent experiments is shown. ( B ) Verification of expression of the wild-type and Hxk2 mutant proteins. The double-mutant strain DBY2052 (Δ hxk1 Δ hxk2GLK1 ) lacking the two fructose-phosphorylating enzymes was used as control. Yeasts were grown to mid-exponential phase in complex medium with galactose, and then shifted to high-glucose medium for 1 h. The cells were harvested and lysed in 1% SDS/PBS containing protease inhibitors. Aliquots of 20 μg of protein/lane were resolved by SDS/12% PAGE. Hxk2 detection was performed using an anti-Hxk2 antibody.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: In Vivo, Binding Assay, Mutagenesis, Gas Chromatography, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Chromatin Immunoprecipitation, Expressing, Polyacrylamide Gel Electrophoresis

    Effect of Hxk2 wca and Hxk2 wrf mutations on glucose repression signalling The strain FMY20VP with the GLK1 gene under the control of the ADH1 constitutive promoter was transformed with plasmids containing HXK2 wca and HXK2 wrf mutant alleles. As controls, we used the FMY20VP strain transformed both with a plasmid containing the wild-type (WT) HXK2 gene and the empty plasmid. We have also included invertase activity of wild-type strain W303.1A. The cells were grown on high-glucose medium (H-Glc, black bars) until a D 600 of 1.0 was reached and then transferred to synthetic medium with low glucose for 60 min (L-Glc, white bars). Invertase activity was assayed in whole cells. Results are means+S.D. of ten measurements obtained with five independent isolated colonies.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Effect of Hxk2 wca and Hxk2 wrf mutations on glucose repression signalling The strain FMY20VP with the GLK1 gene under the control of the ADH1 constitutive promoter was transformed with plasmids containing HXK2 wca and HXK2 wrf mutant alleles. As controls, we used the FMY20VP strain transformed both with a plasmid containing the wild-type (WT) HXK2 gene and the empty plasmid. We have also included invertase activity of wild-type strain W303.1A. The cells were grown on high-glucose medium (H-Glc, black bars) until a D 600 of 1.0 was reached and then transferred to synthetic medium with low glucose for 60 min (L-Glc, white bars). Invertase activity was assayed in whole cells. Results are means+S.D. of ten measurements obtained with five independent isolated colonies.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: Transformation Assay, Mutagenesis, Plasmid Preparation, Activity Assay, Gas Chromatography, Isolation

    Growth assays, catalytic activity and regulatory function of W303.1A wca and W303.1A wrf strains ( A ) The strains W303.1A wca and W303.1A wrf have the HXK2 wca and HXK2 wrf alleles integrated into the HXK2 locus of strain W303.1A respectively. Ten-fold serial dilutions of cultures grown in YEPG were spotted on to complex media containing glucose or galactose and incubated at 28 °C for 48 h. The wild-type (WT) W303.1A strain was used as control. ( B ) The THG1 wca and THG1 wrf strains have the HXK2 wca and HXK2 wrf alleles integrated into the HXK2 locus of strain THG1 respectively. The wild-type W303.1A and the triple mutant strain THG1 (Δ hxk1 Δ hxk2 Δ glk1 ) were used as controls. The cells were grown on YEPG medium until a D 600 of 1.0 was reached and then transferred to YEPD medium for 60 min. Glucose-phosphorylating activity was assayed in crude extracts. Results are means+S.D. of ten measurements obtained with five independent isolated colonies. ( C ) The strains W303.1A wca and W303.1A wrf were grown on YEPG medium until a D 600 of 1.0 was reached and then transferred to high-glucose (H-Glc, black bars) and low-glucose (L-Glc, white bars) complex media for 60 min. Invertase activity was assayed in whole cells. Results are means+S.D. of ten measurements obtained with five independent isolated colonies.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Growth assays, catalytic activity and regulatory function of W303.1A wca and W303.1A wrf strains ( A ) The strains W303.1A wca and W303.1A wrf have the HXK2 wca and HXK2 wrf alleles integrated into the HXK2 locus of strain W303.1A respectively. Ten-fold serial dilutions of cultures grown in YEPG were spotted on to complex media containing glucose or galactose and incubated at 28 °C for 48 h. The wild-type (WT) W303.1A strain was used as control. ( B ) The THG1 wca and THG1 wrf strains have the HXK2 wca and HXK2 wrf alleles integrated into the HXK2 locus of strain THG1 respectively. The wild-type W303.1A and the triple mutant strain THG1 (Δ hxk1 Δ hxk2 Δ glk1 ) were used as controls. The cells were grown on YEPG medium until a D 600 of 1.0 was reached and then transferred to YEPD medium for 60 min. Glucose-phosphorylating activity was assayed in crude extracts. Results are means+S.D. of ten measurements obtained with five independent isolated colonies. ( C ) The strains W303.1A wca and W303.1A wrf were grown on YEPG medium until a D 600 of 1.0 was reached and then transferred to high-glucose (H-Glc, black bars) and low-glucose (L-Glc, white bars) complex media for 60 min. Invertase activity was assayed in whole cells. Results are means+S.D. of ten measurements obtained with five independent isolated colonies.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: Activity Assay, Incubation, Mutagenesis, Isolation, Gas Chromatography

    Yeast two-hybrid interaction of Mig1 with Hxk2 wca and Hxk2 wrf during high-glucose-grown conditions ( A ) Mig1 fused to the GBD were individually co-transformed into yeast strain Y187 with constructs encoding GAD or GAD fused to wild-type Hxk2 and the mutant alleles Hxk2 wca and Hxk2 wrf . The wild-type Hxk2 fused to the GAD was also co-transformed into yeast strain Y187 with the construct encoding the GBD. The transformed yeast cells were grown in SD medium lacking appropriate supplements to maintain selection for plasmids and were harvested at early-exponential phase ( D 600 of 0.6) from high-glucose conditions (H-Glc). Protein–protein interactions were examined in each transformant by the quantitative assay method for β-galactosidase activity. Results are means+S.D. of ten measurements obtained with five independent isolated clones. Strains lacking the reporter constructs did not exhibit any significant activity. ( B ) Western blot analysis of the expression of different HXK2 alleles under high-glucose conditions.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Yeast two-hybrid interaction of Mig1 with Hxk2 wca and Hxk2 wrf during high-glucose-grown conditions ( A ) Mig1 fused to the GBD were individually co-transformed into yeast strain Y187 with constructs encoding GAD or GAD fused to wild-type Hxk2 and the mutant alleles Hxk2 wca and Hxk2 wrf . The wild-type Hxk2 fused to the GAD was also co-transformed into yeast strain Y187 with the construct encoding the GBD. The transformed yeast cells were grown in SD medium lacking appropriate supplements to maintain selection for plasmids and were harvested at early-exponential phase ( D 600 of 0.6) from high-glucose conditions (H-Glc). Protein–protein interactions were examined in each transformant by the quantitative assay method for β-galactosidase activity. Results are means+S.D. of ten measurements obtained with five independent isolated clones. Strains lacking the reporter constructs did not exhibit any significant activity. ( B ) Western blot analysis of the expression of different HXK2 alleles under high-glucose conditions.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: Transformation Assay, Construct, Mutagenesis, Selection, Gas Chromatography, Activity Assay, Isolation, Clone Assay, Western Blot, Expressing

    Interaction of Hxk2 wca and Hxk2 wrf with Snf1 Co-immunoprecipitation of HA–Snf1 with Hxk2 wca and Hxk2 wrf . The FMY303H2 strain, which encoded an HA-tagged Snf1 protein, was transformed with plasmids YEp352/Hxk2, YEp352/Hxk2 wca and YEp352/Hxk2 wrf . The cells were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Snf1 was visualized by Western blotting with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Interaction of Hxk2 wca and Hxk2 wrf with Snf1 Co-immunoprecipitation of HA–Snf1 with Hxk2 wca and Hxk2 wrf . The FMY303H2 strain, which encoded an HA-tagged Snf1 protein, was transformed with plasmids YEp352/Hxk2, YEp352/Hxk2 wca and YEp352/Hxk2 wrf . The cells were grown in SG medium lacking appropriate supplements to maintain selection for plasmid, until a D 600 of 1.0 was reached and then shifted to high- (H-Glc) and low- (L-Glc) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Hxk2 antibody or a polyclonal antibody against Pho4 (lanes 7 and 8). Immunoprecipitates were separated by SDS/12% PAGE, and co-immunoprecipitated HA–Snf1 was visualized by Western blotting with a monoclonal anti-HA antibody. The level of immunoprecipitated Hxk2 in the blotted samples was determined by using an anti-Hxk2 antibody.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: Immunoprecipitation, Transformation Assay, Selection, Plasmid Preparation, Gas Chromatography, Polyacrylamide Gel Electrophoresis, Western Blot

    Growth assays and expression of Hxk2 mutants ( A ) The double-mutant (DM) DBY2052 (Δ hxk1 Δ hxk2 ) strain was transformed with plasmids expressing the indicated HXK2 mutant allele. The strain also contained both the empty vector and the wild-type HXK2 allele as controls. Ten-fold serial dilutions of cultures grown in selective medium with galactose were spotted on to selective media containing fructose or glucose and incubated at 28 °C for 48 h. The mutants, listed on the left, are arranged in the order in which they occur in the primary sequence of Hxk2. The first two rows show wild-type (WT, W303.1A) and the double-mutant Δ hxk1 Δ hxk2 strain transformed with the empty vector (YEp352) as controls. The last two rows show the catalytic mutant allele HXK2 wca and the regulatory mutant allele HXK2 wrf . ( B ) Verification of expression of the wild-type and Hxk2 mutant proteins. The double-mutant strain DBY2052 (Δ hxk1 Δ hxk2GLK1 ) lacking the two fructose-phosphorylating enzymes was transformed with the multicopy plasmids indicated. Yeasts were grown to the mid-exponential phase in selective medium with galactose and then shifted to high-glucose medium for 1 h, harvested and lysed in 1% SDS/PBS containing protease inhibitors. Aliquots of 20 μg of protein/lane were resolved by SDS/12% PAGE. Hxk2 detection was performed using an anti-Hxk2 antibody.

    Journal: Biochemical Journal

    Article Title: Functional domains of yeast hexokinase 2

    doi: 10.1042/BJ20100663

    Figure Lengend Snippet: Growth assays and expression of Hxk2 mutants ( A ) The double-mutant (DM) DBY2052 (Δ hxk1 Δ hxk2 ) strain was transformed with plasmids expressing the indicated HXK2 mutant allele. The strain also contained both the empty vector and the wild-type HXK2 allele as controls. Ten-fold serial dilutions of cultures grown in selective medium with galactose were spotted on to selective media containing fructose or glucose and incubated at 28 °C for 48 h. The mutants, listed on the left, are arranged in the order in which they occur in the primary sequence of Hxk2. The first two rows show wild-type (WT, W303.1A) and the double-mutant Δ hxk1 Δ hxk2 strain transformed with the empty vector (YEp352) as controls. The last two rows show the catalytic mutant allele HXK2 wca and the regulatory mutant allele HXK2 wrf . ( B ) Verification of expression of the wild-type and Hxk2 mutant proteins. The double-mutant strain DBY2052 (Δ hxk1 Δ hxk2GLK1 ) lacking the two fructose-phosphorylating enzymes was transformed with the multicopy plasmids indicated. Yeasts were grown to the mid-exponential phase in selective medium with galactose and then shifted to high-glucose medium for 1 h, harvested and lysed in 1% SDS/PBS containing protease inhibitors. Aliquots of 20 μg of protein/lane were resolved by SDS/12% PAGE. Hxk2 detection was performed using an anti-Hxk2 antibody.

    Article Snippet: Extracts were incubated with anti-Hxk2 antibody (Santa Cruz Biotechnology) or anti-Pho4 antibody for 1 h at 4 °C.

    Techniques: Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation, Incubation, Sequencing, Polyacrylamide Gel Electrophoresis

    Interaction of Kap60 and Xpo1 with Hxk2 variants. A and B , in vivo co-immunoprecipitation of Kap60 with Hxk2, Hxk2S14A, and Hxk2S14D is shown. The W303-1A (WT), FMY308, and FMY309 strains were grown in YEPD medium until an A 600 nm of 0.8 was reached and then shifted to high ( H-Glc ) and low ( L-Glc ) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Kap60 antibody ( lanes 1–4 ) or a polyclonal antibody to Pho4 ( lanes 5 and 6 ). Immunoprecipitates were separated by 12% SDS-PAGE, and co-precipitated Hxk2 variants were visualized on a Western blot with monoclonal anti-Hxk2 antibody. The level of immunoprecipitated Kap60 in the blotted samples was determined by using anti-Kap60 antibody. The level of Hxk2 present in the different extracts was determined by Western blot using anti-Hxk2 antibody. C and D , GST pulldown assay of the interaction between Xpo1 and Hxk2 variants. The GST-Xpo1 fusion protein was purified using glutathione-Sepharose columns. Equal amounts of GST-Xpo1 were incubated with cell extracts from the W303-1A (WT), FMY308, and FMY309 strains. The yeast strains were grown in YEPD medium until an A 600 nm of 0.8 was reached and then shifted to low glucose conditions for 1 h. After exhaustive washing, the proteins were separated by 12% SDS-PAGE, and retained Hxk2 variants were visualized on a Western blot with polyclonal anti-Hxk2 antibody ( lanes 1–4 ). For the control samples, GST protein was also incubated with the high and low glucose cell extracts, respectively, but no signals were detected ( lanes 5 and 6 ). The level of Hxk2 present in the different extracts used in C and D was determined by Western blot using anti-Hxk2 antibody. All Western blots shown are representative of results obtained from four independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling *

    doi: 10.1074/jbc.M112.401679

    Figure Lengend Snippet: Interaction of Kap60 and Xpo1 with Hxk2 variants. A and B , in vivo co-immunoprecipitation of Kap60 with Hxk2, Hxk2S14A, and Hxk2S14D is shown. The W303-1A (WT), FMY308, and FMY309 strains were grown in YEPD medium until an A 600 nm of 0.8 was reached and then shifted to high ( H-Glc ) and low ( L-Glc ) glucose conditions for 1 h. The cell extracts were immunoprecipitated with a polyclonal anti-Kap60 antibody ( lanes 1–4 ) or a polyclonal antibody to Pho4 ( lanes 5 and 6 ). Immunoprecipitates were separated by 12% SDS-PAGE, and co-precipitated Hxk2 variants were visualized on a Western blot with monoclonal anti-Hxk2 antibody. The level of immunoprecipitated Kap60 in the blotted samples was determined by using anti-Kap60 antibody. The level of Hxk2 present in the different extracts was determined by Western blot using anti-Hxk2 antibody. C and D , GST pulldown assay of the interaction between Xpo1 and Hxk2 variants. The GST-Xpo1 fusion protein was purified using glutathione-Sepharose columns. Equal amounts of GST-Xpo1 were incubated with cell extracts from the W303-1A (WT), FMY308, and FMY309 strains. The yeast strains were grown in YEPD medium until an A 600 nm of 0.8 was reached and then shifted to low glucose conditions for 1 h. After exhaustive washing, the proteins were separated by 12% SDS-PAGE, and retained Hxk2 variants were visualized on a Western blot with polyclonal anti-Hxk2 antibody ( lanes 1–4 ). For the control samples, GST protein was also incubated with the high and low glucose cell extracts, respectively, but no signals were detected ( lanes 5 and 6 ). The level of Hxk2 present in the different extracts used in C and D was determined by Western blot using anti-Hxk2 antibody. All Western blots shown are representative of results obtained from four independent experiments.

    Article Snippet: The extracts were incubated with anti-Kap60, anti-Hxk2, or anti-Pho4 polyclonal antibody for 3 h at 4 °C.

    Techniques: In Vivo, Immunoprecipitation, Gas Chromatography, SDS Page, Western Blot, GST Pulldown Assay, Purification, Incubation

    Localization of Hxk2S14A and Hxk2S14D in Δ hxk1 Δ hxk2 yeast cells. The DBY2052 (Δ hxk1 Δ hxk2 ) mutant strain was transformed with plasmids YEp352/Hxk2-GFP, YEp352/Hxk2S14A-GFP ( A ), YEp352/Hxk2nes2-GFP, and YEp352/Hxk2 nes2 S14D-GFP ( B ) plasmids. Transformed cells were grown in high glucose synthetic medium ( H-Glc ) until an A 600 nm of 1.0 was reached and then transferred to low glucose synthetic medium ( L-Glc ) for 60 min. The cells were visualized by fluorescence microscopy; DAPI staining revealed nuclear DNA. The nuclear localization of fluorescent reporter proteins was determined in at least 100 cells in three independent experiments. Means and S.D. ( error bars ) are shown for at least three independent experiments. *, statistically significant differences of Hxk2S14A versus Hxk2 in low glucose conditions, p

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling *

    doi: 10.1074/jbc.M112.401679

    Figure Lengend Snippet: Localization of Hxk2S14A and Hxk2S14D in Δ hxk1 Δ hxk2 yeast cells. The DBY2052 (Δ hxk1 Δ hxk2 ) mutant strain was transformed with plasmids YEp352/Hxk2-GFP, YEp352/Hxk2S14A-GFP ( A ), YEp352/Hxk2nes2-GFP, and YEp352/Hxk2 nes2 S14D-GFP ( B ) plasmids. Transformed cells were grown in high glucose synthetic medium ( H-Glc ) until an A 600 nm of 1.0 was reached and then transferred to low glucose synthetic medium ( L-Glc ) for 60 min. The cells were visualized by fluorescence microscopy; DAPI staining revealed nuclear DNA. The nuclear localization of fluorescent reporter proteins was determined in at least 100 cells in three independent experiments. Means and S.D. ( error bars ) are shown for at least three independent experiments. *, statistically significant differences of Hxk2S14A versus Hxk2 in low glucose conditions, p

    Article Snippet: The extracts were incubated with anti-Kap60, anti-Hxk2, or anti-Pho4 polyclonal antibody for 3 h at 4 °C.

    Techniques: Mutagenesis, Transformation Assay, Gas Chromatography, Fluorescence, Microscopy, Staining

    Structural analysis of the Hxk2 NLS region. Electrostatic surface presentations of Hxk2S14A ( A ) and Hxk2S14D ( B ) are shown. The NLS region ( circled ) displays increased electronegativity due to the S14D mutation, which could have an effect on Xpo1 recognition. C , the distances between the Cα of different residues (His-2 to Asp-17 and Lys-6 to Val-375) from Hxk2S14A and Hxk2S14D are shown; they suggest a significant conformational change in the NLS region. D , the distances between the Cα of different residues (Val-1 to Leu-22 and Val-1 to Met-318) from Hxk2S14A and Hxk2S14D are shown; they suggest that the conformational change in the NLS region introduces Val-1 between the two NES sequences.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling *

    doi: 10.1074/jbc.M112.401679

    Figure Lengend Snippet: Structural analysis of the Hxk2 NLS region. Electrostatic surface presentations of Hxk2S14A ( A ) and Hxk2S14D ( B ) are shown. The NLS region ( circled ) displays increased electronegativity due to the S14D mutation, which could have an effect on Xpo1 recognition. C , the distances between the Cα of different residues (His-2 to Asp-17 and Lys-6 to Val-375) from Hxk2S14A and Hxk2S14D are shown; they suggest a significant conformational change in the NLS region. D , the distances between the Cα of different residues (Val-1 to Leu-22 and Val-1 to Met-318) from Hxk2S14A and Hxk2S14D are shown; they suggest that the conformational change in the NLS region introduces Val-1 between the two NES sequences.

    Article Snippet: The extracts were incubated with anti-Kap60, anti-Hxk2, or anti-Pho4 polyclonal antibody for 3 h at 4 °C.

    Techniques: Mutagenesis

    CYGB suppressed xenograft tumor growth in vivo. a Image of xenografts before and after resection. Red and black arrows indicate empty vector control tumors and CYGB-overexpressing tumors, respectively. b Tumor weight (4 xenografts/group). c Growth curve of xenograft tumors. d Representative images of immunohistochemistry (IHC) staining. Paraffin sections were prepared from the tumors of mice #2 and stained for CYGB, GLUT1, HXK2, and TIGAR. *: p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The epigenetically downregulated factor CYGB suppresses breast cancer through inhibition of glucose metabolism

    doi: 10.1186/s13046-018-0979-9

    Figure Lengend Snippet: CYGB suppressed xenograft tumor growth in vivo. a Image of xenografts before and after resection. Red and black arrows indicate empty vector control tumors and CYGB-overexpressing tumors, respectively. b Tumor weight (4 xenografts/group). c Growth curve of xenograft tumors. d Representative images of immunohistochemistry (IHC) staining. Paraffin sections were prepared from the tumors of mice #2 and stained for CYGB, GLUT1, HXK2, and TIGAR. *: p

    Article Snippet: The following primary antibodies were used in this study: CYGB (Santa Cruz Biotechnology, sc-365,246), GAPDH (Cell Signaling Technology, #2118), p53 (Santa Cruz Biotechnology, sc-126), p21 (Cell Signaling Technology, #2947), β-actin (Abcam, ab8226), GLUT1 (Santa Cruz Biotechnology, sc-377,228), HXK2 (Santa Cruz Biotechnology, sc-374,091), TIGAR (Santa Cruz Biotechnology, sc-166,290).

    Techniques: In Vivo, Plasmid Preparation, Immunohistochemistry, Staining, Mouse Assay

    Induction of Glut1 and HXK2 in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p

    Journal: Scientific Reports

    Article Title: TRAF3 deficiency promotes metabolic reprogramming in B cells

    doi: 10.1038/srep35349

    Figure Lengend Snippet: Induction of Glut1 and HXK2 in B cells in the absence of TRAF3. ( A,B ) B cells were isolated from littermate WT and B- Traf3 −/− mice. ( A ) Whole cell lysates were analyzed with Western blotting (WB) for Glut1 and HXK2 expression. Band intensities were quantified and normalized to actin. Graphs depict mean values ± SEM from three independent experiments. Full-length blots are presented in Supplementary Fig. S4 . ( B ) Glut1 and Hxk2 mRNA levels were assayed with RT-PCR and analyzed as described in Materials and Methods. Data were normalized to GAPDH and fold change was determined using the comparative Ct method. N = 3 mice with mean values ± SEM shown. Student’s t test was used to evaluate differences for statistical significance in A and B (*p

    Article Snippet: Antibodies and reagents Anti-Glut1 rabbit mAb (ab652) was purchased from Abcam (Cambridge, MA) and anti-HXK2 rabbit mAb (C64G5) from Cell Signaling (Danvers, MA).

    Techniques: Isolation, Mouse Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction