R32803 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • N/A
    Glutathione S-transferases pi (GSTP1), also known as GST3, is an enzyme that in humans is encoded by the GSTP1 gene. This gene is mapped to 11q13.2. GSTP1 has 7 exons and 6 introns contained within
      Buy from Supplier

    86
    Abiocode Inc anti bri1
    Glucose influences the interactions between <t>BRI1</t> and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test
    Anti Bri1, supplied by Abiocode Inc, 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 bri1/product/Abiocode Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti bri1 - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier

    86
    Millipore anti rpa32
    ( A ) Stable U2-OS cells expressing WT- or <t>PD-RPA32</t> were treated with 10 uM CPT for 3 hrs to induce RPA hyperphosphorylation. Cells were harvested and chromatin-bound proteins were isolated. Chromatin was subjected to DNase I digestion and 10% of the sample was loaded onto the gel (INPUT). The remaining lysate was immunoprecipitated using anti-p53 antibody. Samples were analyzed by Western blotting. ( B ) Immunoprecipitation was performed with A549 cell lysate using anti-p53 antibody. Immunoprecipitates were washed with buffer of increasing salt concentrations (0 – 1.0 M) to remove proteins bound to p53, including RPA and Mdm2. Samples were then analyzed by Western blotting. ( C ) p53 from A549 cell lysates treated with 10 uM CPT for 2 hrs or 2 mM HU for 24 hrs was isolated by IP with anti-p53 antibody, followed by a 1 M salt buffer wash. Equimolar amounts of purified RPA and hyp-RPA were added and the proteins were allowed to interact for 6 hrs. Then, the p53 complexes were pulled down, washed and analyzed by Western blotting.
    Anti Rpa32, supplied by Millipore, 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 rpa32/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti rpa32 - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier

    86
    Millipore anti bri1
    Glucose influences the interactions between <t>BRI1</t> and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test
    Anti Bri1, supplied by Millipore, 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 bri1/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti bri1 - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier


    Image Search Results


    Glucose influences the interactions between BRI1 and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: Glucose influences the interactions between BRI1 and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques: Incubation, Immunoprecipitation, Western Blot

    G-proteins act genetically with BRI1 and BAK1. a Comparison of development stages of Col-0 (1), bri1-301 (2), bak1-4 (3), agb1-2 (4), agb1-2 bri1-301 (5), agb1-2 bak1-4 (6), gpa1-101 (7), gpa1-101 bri1-301 (8), gpa1-101 bak1-4 (9), agg3-3 (10), agg3-3 bri1-301 , (11), and agg3-3 bak1-4 (12). Seedlings were grown vertically on medium supplemented with 1% glucose in the dark for 19 days ( n ≥ 85). b The percentage of the indicated seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose under constant light condition for 16 days ( n ≥ 72). Values in a.b are given as mean ± SD

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: G-proteins act genetically with BRI1 and BAK1. a Comparison of development stages of Col-0 (1), bri1-301 (2), bak1-4 (3), agb1-2 (4), agb1-2 bri1-301 (5), agb1-2 bak1-4 (6), gpa1-101 (7), gpa1-101 bri1-301 (8), gpa1-101 bak1-4 (9), agg3-3 (10), agg3-3 bri1-301 , (11), and agg3-3 bak1-4 (12). Seedlings were grown vertically on medium supplemented with 1% glucose in the dark for 19 days ( n ≥ 85). b The percentage of the indicated seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose under constant light condition for 16 days ( n ≥ 72). Values in a.b are given as mean ± SD

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques:

    BRI1 and BAK1 physically interact with G proteins. a BRI1 kinase domain (BRI1-KD) interacts with AGB1 and AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BRI1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1.IN, input; IB, immunoblot. b BAK1 kinase domain (BAK1-KD) interacts with AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BAK1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1. IN, input; IB immunoblot. c Bimolecular fluorescence complementation (BiFC) assays show that BRI1 interacts with AGB1 and AGG3 in N . benthamiana leaves. BRI1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . d BiFC assays show that BAK1 interacts with AGG3 in N . benthamiana leaves. BAK1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . e BRI1 interacts with AGB1 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:Myc-AGB1 , or pBRI1:BRI1-GFP ; 35S:Myc-AGB1 seedlings were incubated with GFP-Trap-A, respectively. Immunoprecipitates were examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. f BRI1 interacts with AGG3 in Arabidopsis. The extracted total proteins from pBRI1:BRI1-GFP ; 35S:GFP or pBRI1:BRI1-GFP ; 35S:Myc-AGG3 seedlings were incubated with GFP-Trap-A, and examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. g BAK1 interacts with AGG3 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:BAK1-HA and 35S:GFP-AGG3 ; 35S:BAK1-HA seedlings were incubated with GFP-Trap-A, and examined using anti-HA and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. Scale bars, 50 µm in c , d

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 physically interact with G proteins. a BRI1 kinase domain (BRI1-KD) interacts with AGB1 and AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BRI1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1.IN, input; IB, immunoblot. b BAK1 kinase domain (BAK1-KD) interacts with AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BAK1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1. IN, input; IB immunoblot. c Bimolecular fluorescence complementation (BiFC) assays show that BRI1 interacts with AGB1 and AGG3 in N . benthamiana leaves. BRI1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . d BiFC assays show that BAK1 interacts with AGG3 in N . benthamiana leaves. BAK1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . e BRI1 interacts with AGB1 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:Myc-AGB1 , or pBRI1:BRI1-GFP ; 35S:Myc-AGB1 seedlings were incubated with GFP-Trap-A, respectively. Immunoprecipitates were examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. f BRI1 interacts with AGG3 in Arabidopsis. The extracted total proteins from pBRI1:BRI1-GFP ; 35S:GFP or pBRI1:BRI1-GFP ; 35S:Myc-AGG3 seedlings were incubated with GFP-Trap-A, and examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. g BAK1 interacts with AGG3 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:BAK1-HA and 35S:GFP-AGG3 ; 35S:BAK1-HA seedlings were incubated with GFP-Trap-A, and examined using anti-HA and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. Scale bars, 50 µm in c , d

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques: In Vitro, Western Blot, Fluorescence, Incubation, Immunoprecipitation

    BRI1 is required for the phosphorylation of AGB1 and AGG3. a BRI1 kinase domain (BRI1-KD) phosphorylates AGB1 in vitro, but bri1-301 kinase domain (bri1-301-KD) does not. The phosphorylated MBP-AGB1 (pMBP-AGB1), unphosphorylated (MBP-AGB1) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. b BRI1-KD phosphorylates AGG3 in vitro, but bri1-301-KD does not. The phosphorylated MBP-AGG3 (pMBP-AGG3), unphosphorylated (MBP-AGG3) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. c , d BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGB1 (L1) and 35S:GFP ; 35S:Myc-AGB1 (L2) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively c . Quantification of the phosphorylated Myc-AGB1 was relative to the immunoprecipitated Myc-AGB1 d . Values are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for L2 seedlings by Student’s t -test. e , f BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGG3 (L3) and 35S:GFP ; 35S:Myc-AGG3 (L4) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively ( e ). Quantification of the phosphorylated Myc-AGG3 was relative to the immunoprecipitated Myc-AGG3 f . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L4 seedlings by Student’s t -test. g , h BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from 35S:GFP-AGB1 (L5) and 35S:GFP-AGB1 ; bri1-301 (L6) seedlings were incubated with GFP-Trap-A, and detected using anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively g . Quantification of the phosphorylated GFP-AGB1 was relative to the immunoprecipitated GFP-AGB1 h . Values are given as mean ± SD, ( n = 3).** P < 0.01 compared with the value for L5 seedlings by Student’s t -test. i , j BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from 35S:GFP-AGG3 (L7) and 35S:GFP-AGG3 ; bri1-301 (L8) seedlings were incubated with GFP beads, and detected by immunoblot with anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively i . Quantification of the phosphorylated GFP-AGG3 was relative to the immunoprecipitated GFP-AGG3 j . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L7 seedlings by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 is required for the phosphorylation of AGB1 and AGG3. a BRI1 kinase domain (BRI1-KD) phosphorylates AGB1 in vitro, but bri1-301 kinase domain (bri1-301-KD) does not. The phosphorylated MBP-AGB1 (pMBP-AGB1), unphosphorylated (MBP-AGB1) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. b BRI1-KD phosphorylates AGG3 in vitro, but bri1-301-KD does not. The phosphorylated MBP-AGG3 (pMBP-AGG3), unphosphorylated (MBP-AGG3) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. c , d BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGB1 (L1) and 35S:GFP ; 35S:Myc-AGB1 (L2) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively c . Quantification of the phosphorylated Myc-AGB1 was relative to the immunoprecipitated Myc-AGB1 d . Values are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for L2 seedlings by Student’s t -test. e , f BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGG3 (L3) and 35S:GFP ; 35S:Myc-AGG3 (L4) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively ( e ). Quantification of the phosphorylated Myc-AGG3 was relative to the immunoprecipitated Myc-AGG3 f . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L4 seedlings by Student’s t -test. g , h BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from 35S:GFP-AGB1 (L5) and 35S:GFP-AGB1 ; bri1-301 (L6) seedlings were incubated with GFP-Trap-A, and detected using anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively g . Quantification of the phosphorylated GFP-AGB1 was relative to the immunoprecipitated GFP-AGB1 h . Values are given as mean ± SD, ( n = 3).** P < 0.01 compared with the value for L5 seedlings by Student’s t -test. i , j BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from 35S:GFP-AGG3 (L7) and 35S:GFP-AGG3 ; bri1-301 (L8) seedlings were incubated with GFP beads, and detected by immunoblot with anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively i . Quantification of the phosphorylated GFP-AGG3 was relative to the immunoprecipitated GFP-AGG3 j . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L7 seedlings by Student’s t -test

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques: In Vitro, SDS Page, Immunoprecipitation, Incubation, Western Blot

    BRI1 and BAK1 affect the interactions between GPA1 and AGB1. a BRI1 and BAK1 influences the interactions between GPA1 and AGB1 in Arabidopsis , respectively. The wild type, bri1-301 , and bak1-4 leaf protoplasts co-transformed with 35S:AGB1 - Myc and 35S : GPA1 - HA plasmids were grown in darkness for 14 h, and then treated without or with 2% glucose (G) for 5 h. Total proteins from these leaf protoplasts were incubated with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-HA antibodies, respectively. IN input; IP immunoprecipitation. b – d Quantification of relative interactions between GPA1 and AGB1 in wild type ( b ), bri1-301 ( c ) and bak1-4 ( d ) leaf protoplasts treated without or with 2% glucose as shown in a . The ratio was shown as immunoprecipitated GPA1-HA to input GPA1-HA. Values are given as mean ± SD ( n = 3) relative to the value for 0% glucose, set at 1. Values in b – d are given as mean ± SD, ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 affect the interactions between GPA1 and AGB1. a BRI1 and BAK1 influences the interactions between GPA1 and AGB1 in Arabidopsis , respectively. The wild type, bri1-301 , and bak1-4 leaf protoplasts co-transformed with 35S:AGB1 - Myc and 35S : GPA1 - HA plasmids were grown in darkness for 14 h, and then treated without or with 2% glucose (G) for 5 h. Total proteins from these leaf protoplasts were incubated with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-HA antibodies, respectively. IN input; IP immunoprecipitation. b – d Quantification of relative interactions between GPA1 and AGB1 in wild type ( b ), bri1-301 ( c ) and bak1-4 ( d ) leaf protoplasts treated without or with 2% glucose as shown in a . The ratio was shown as immunoprecipitated GPA1-HA to input GPA1-HA. Values are given as mean ± SD ( n = 3) relative to the value for 0% glucose, set at 1. Values in b – d are given as mean ± SD, ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques: Transformation Assay, Incubation, Immunoprecipitation

    BRI1 and BAK1 play key roles in sugar responses. a Different stages of dark-grown wild-type seedlings used for scoring sugar responses. b Comparison of developmental stages between Col-0, bri1-301 , and bak1-4 . Seedlings were grown vertically on medium supplemented with 1% glucose (G) or 1% mannitol (M) in the dark for 19 days ( n ≥ 83). c The percentage of Col-0, bri1-301 , and bak1-4 seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose (G) or 6% mannitol (M) under constant light condition for 10 days ( n ≥ 89). Values in b , c are given as mean ± SD. ** P < 0.01 compared with the wild type by Student’s t -test. Scale bars, 2 mm ( a )

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 play key roles in sugar responses. a Different stages of dark-grown wild-type seedlings used for scoring sugar responses. b Comparison of developmental stages between Col-0, bri1-301 , and bak1-4 . Seedlings were grown vertically on medium supplemented with 1% glucose (G) or 1% mannitol (M) in the dark for 19 days ( n ≥ 83). c The percentage of Col-0, bri1-301 , and bak1-4 seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose (G) or 6% mannitol (M) under constant light condition for 10 days ( n ≥ 89). Values in b , c are given as mean ± SD. ** P < 0.01 compared with the wild type by Student’s t -test. Scale bars, 2 mm ( a )

    Article Snippet: To examine the phosphorylation levels of BRI1 and BAK1 using their native antibodies, wild-type seedings were isolated with the extraction buffer, and incubated with anti-BRI1 (Abiocode R3283-4, 1/500) or anti-BAK1 (Abiocode R2350-3, 1/500) antibodies for 30 min, then incubated with Protein G Magnetic Beads for 30 min to immunoprecipitate BRI1 and BAK1, respectively.

    Techniques:

    ( A ) Stable U2-OS cells expressing WT- or PD-RPA32 were treated with 10 uM CPT for 3 hrs to induce RPA hyperphosphorylation. Cells were harvested and chromatin-bound proteins were isolated. Chromatin was subjected to DNase I digestion and 10% of the sample was loaded onto the gel (INPUT). The remaining lysate was immunoprecipitated using anti-p53 antibody. Samples were analyzed by Western blotting. ( B ) Immunoprecipitation was performed with A549 cell lysate using anti-p53 antibody. Immunoprecipitates were washed with buffer of increasing salt concentrations (0 – 1.0 M) to remove proteins bound to p53, including RPA and Mdm2. Samples were then analyzed by Western blotting. ( C ) p53 from A549 cell lysates treated with 10 uM CPT for 2 hrs or 2 mM HU for 24 hrs was isolated by IP with anti-p53 antibody, followed by a 1 M salt buffer wash. Equimolar amounts of purified RPA and hyp-RPA were added and the proteins were allowed to interact for 6 hrs. Then, the p53 complexes were pulled down, washed and analyzed by Western blotting.

    Journal: Oncogene

    Article Title: DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair

    doi: 10.1038/onc.2012.257

    Figure Lengend Snippet: ( A ) Stable U2-OS cells expressing WT- or PD-RPA32 were treated with 10 uM CPT for 3 hrs to induce RPA hyperphosphorylation. Cells were harvested and chromatin-bound proteins were isolated. Chromatin was subjected to DNase I digestion and 10% of the sample was loaded onto the gel (INPUT). The remaining lysate was immunoprecipitated using anti-p53 antibody. Samples were analyzed by Western blotting. ( B ) Immunoprecipitation was performed with A549 cell lysate using anti-p53 antibody. Immunoprecipitates were washed with buffer of increasing salt concentrations (0 – 1.0 M) to remove proteins bound to p53, including RPA and Mdm2. Samples were then analyzed by Western blotting. ( C ) p53 from A549 cell lysates treated with 10 uM CPT for 2 hrs or 2 mM HU for 24 hrs was isolated by IP with anti-p53 antibody, followed by a 1 M salt buffer wash. Equimolar amounts of purified RPA and hyp-RPA were added and the proteins were allowed to interact for 6 hrs. Then, the p53 complexes were pulled down, washed and analyzed by Western blotting.

    Article Snippet: Antibodies used in this study include anti-RPA32 (Sigma R3280), anti-p53 (Invitrogen AHO0142 or Santa Cruz sc-6243), anti-phospho-p53(pSer15) (R&D AF1043 and Cell Signaling 9286), anti-phospho-p53(pSer20) (AnaSpec 54428), anti-phospho-p53(pSer37) (Santa Cruz sc-135633), anti-phospho-p53(Ser46) (Cell Signaling 2521), anti-DNA-PK (Santa Cruz sc-9051), anti-ATM (Bethyl Lab A300-299A), anti-ATR (Bethyl Lab A300-138A) and anti-RAD51 (Santa Cruz sc-8349).

    Techniques: Expressing, Isolation, Immunoprecipitation, Western Blot, Purification

    ( A ) Stable U2OS cells expressing WT- or PD-RPA32 were treated with CPT in a dose-dependent manner for 2 hrs. Comet assay under neutral conditions was performed to assess the efficiency of DSB repair. ( B ) Tail moment was measured using the Comet Assay IV software (Perceptive). At least 50 cells were assessed per treatment (* represents a p-value less than 0.001). ( C ) Co-immunoprecipitation assay was performed simultaneously using duplicate cell cultures. Nuclear lysates were isolated and anti-p53 antibody was used for immunoprecipitation; samples were then analyzed by Western blotting using the indicated antibodies. ( D ) A549 cells were treated with CPT or mock treated, followed by nuclear fractionation and incubation with DNase I. Soluble fractions were incubated with anti-p53 antibodies for co-immunoprecipitation (lanes 3–4). The supernatant after p53 IP then was immunoprecipitated again using Rad51 antibodies (lanes 7–8). ( E ) Cells expressing WT- or PD-RPA32 were treated with CPT and subjected to immunofluorescence microscopic determination of nuclear focus formation of Rad52. ( F ) Quantitative analysis of the data from ( E ). 100 cells were randomly selected in three separate experiments. Cells with at least one focus were counted (* represents a p-value less than 0.001).

    Journal: Oncogene

    Article Title: DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair

    doi: 10.1038/onc.2012.257

    Figure Lengend Snippet: ( A ) Stable U2OS cells expressing WT- or PD-RPA32 were treated with CPT in a dose-dependent manner for 2 hrs. Comet assay under neutral conditions was performed to assess the efficiency of DSB repair. ( B ) Tail moment was measured using the Comet Assay IV software (Perceptive). At least 50 cells were assessed per treatment (* represents a p-value less than 0.001). ( C ) Co-immunoprecipitation assay was performed simultaneously using duplicate cell cultures. Nuclear lysates were isolated and anti-p53 antibody was used for immunoprecipitation; samples were then analyzed by Western blotting using the indicated antibodies. ( D ) A549 cells were treated with CPT or mock treated, followed by nuclear fractionation and incubation with DNase I. Soluble fractions were incubated with anti-p53 antibodies for co-immunoprecipitation (lanes 3–4). The supernatant after p53 IP then was immunoprecipitated again using Rad51 antibodies (lanes 7–8). ( E ) Cells expressing WT- or PD-RPA32 were treated with CPT and subjected to immunofluorescence microscopic determination of nuclear focus formation of Rad52. ( F ) Quantitative analysis of the data from ( E ). 100 cells were randomly selected in three separate experiments. Cells with at least one focus were counted (* represents a p-value less than 0.001).

    Article Snippet: Antibodies used in this study include anti-RPA32 (Sigma R3280), anti-p53 (Invitrogen AHO0142 or Santa Cruz sc-6243), anti-phospho-p53(pSer15) (R&D AF1043 and Cell Signaling 9286), anti-phospho-p53(pSer20) (AnaSpec 54428), anti-phospho-p53(pSer37) (Santa Cruz sc-135633), anti-phospho-p53(Ser46) (Cell Signaling 2521), anti-DNA-PK (Santa Cruz sc-9051), anti-ATM (Bethyl Lab A300-299A), anti-ATR (Bethyl Lab A300-138A) and anti-RAD51 (Santa Cruz sc-8349).

    Techniques: Expressing, Single Cell Gel Electrophoresis, Software, Co-Immunoprecipitation Assay, Isolation, Immunoprecipitation, Western Blot, Fractionation, Incubation, Immunofluorescence

    Glucose influences the interactions between BRI1 and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: Glucose influences the interactions between BRI1 and BAK1. a Glucose regulates the physical interactions between BRI1 and BAK1. pBRI1:BRI1-GFP ; 35S:BAK1 - HA or 35S:BAK1-HA seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. b Quantification of BRI1-GFP and BAK1-HA associations in Arabidopsis seedlings treated with different concentrations of glucose (G) for 4 h from a . The ratio value of immunoprecipitated BAK1-HA to input BAK1-HA in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. c Glucose influences the phosphorylation levels of BRI1 in Arabidopsis. pBRI1:BRI1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN input; IP immunoprecipitation; IB immunoblot. d Quantification of BRI1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from c . The ratio value of phosphorylated BRI1-GFP to immunoprecipitated BRI1-GFP in response to 0% glucose was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test. e Glucose influences the phosphorylation levels of BAK1 in Arabidopsis. pBAK1:BAK1-GFP seedlings were grown in the light for 5 days, then incubated in darkness for 4 days, and treated with different concentrations of glucose (G) for 4 or 24 h. IN, input; IP immunoprecipitation; IB immunoblot. f Quantification of BAK1-GFP phosphorylation in Arabidopsis seedlings treated with different concentrations of glucose for 4 h from e . The ratio value of phosphorylated BAK1-GFP to immunoprecipitated BAK1-GFP was set at 1. Values for glucose treatments are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques: Incubation, Immunoprecipitation, Western Blot

    G-proteins act genetically with BRI1 and BAK1. a Comparison of development stages of Col-0 (1), bri1-301 (2), bak1-4 (3), agb1-2 (4), agb1-2 bri1-301 (5), agb1-2 bak1-4 (6), gpa1-101 (7), gpa1-101 bri1-301 (8), gpa1-101 bak1-4 (9), agg3-3 (10), agg3-3 bri1-301 , (11), and agg3-3 bak1-4 (12). Seedlings were grown vertically on medium supplemented with 1% glucose in the dark for 19 days ( n ≥ 85). b The percentage of the indicated seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose under constant light condition for 16 days ( n ≥ 72). Values in a.b are given as mean ± SD

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: G-proteins act genetically with BRI1 and BAK1. a Comparison of development stages of Col-0 (1), bri1-301 (2), bak1-4 (3), agb1-2 (4), agb1-2 bri1-301 (5), agb1-2 bak1-4 (6), gpa1-101 (7), gpa1-101 bri1-301 (8), gpa1-101 bak1-4 (9), agg3-3 (10), agg3-3 bri1-301 , (11), and agg3-3 bak1-4 (12). Seedlings were grown vertically on medium supplemented with 1% glucose in the dark for 19 days ( n ≥ 85). b The percentage of the indicated seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose under constant light condition for 16 days ( n ≥ 72). Values in a.b are given as mean ± SD

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques:

    BRI1 and BAK1 physically interact with G proteins. a BRI1 kinase domain (BRI1-KD) interacts with AGB1 and AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BRI1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1.IN, input; IB, immunoblot. b BAK1 kinase domain (BAK1-KD) interacts with AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BAK1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1. IN, input; IB immunoblot. c Bimolecular fluorescence complementation (BiFC) assays show that BRI1 interacts with AGB1 and AGG3 in N . benthamiana leaves. BRI1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . d BiFC assays show that BAK1 interacts with AGG3 in N . benthamiana leaves. BAK1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . e BRI1 interacts with AGB1 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:Myc-AGB1 , or pBRI1:BRI1-GFP ; 35S:Myc-AGB1 seedlings were incubated with GFP-Trap-A, respectively. Immunoprecipitates were examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. f BRI1 interacts with AGG3 in Arabidopsis. The extracted total proteins from pBRI1:BRI1-GFP ; 35S:GFP or pBRI1:BRI1-GFP ; 35S:Myc-AGG3 seedlings were incubated with GFP-Trap-A, and examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. g BAK1 interacts with AGG3 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:BAK1-HA and 35S:GFP-AGG3 ; 35S:BAK1-HA seedlings were incubated with GFP-Trap-A, and examined using anti-HA and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. Scale bars, 50 µm in c , d

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 physically interact with G proteins. a BRI1 kinase domain (BRI1-KD) interacts with AGB1 and AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BRI1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1.IN, input; IB, immunoblot. b BAK1 kinase domain (BAK1-KD) interacts with AGG3 in vitro. MBP-GPA1, MBP-AGB1, or MBP-AGG3 was pulled down by GST-BAK1-KD and detected by immunoblot with anti-MBP antibody. Arrow indicates the band of MBP-GPA1. IN, input; IB immunoblot. c Bimolecular fluorescence complementation (BiFC) assays show that BRI1 interacts with AGB1 and AGG3 in N . benthamiana leaves. BRI1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . d BiFC assays show that BAK1 interacts with AGG3 in N . benthamiana leaves. BAK1-nYFP was coexpressed with GPA1-cYFP, AGB1-cYFP, or AGG3-cYFP in leaves of N . benthamiana . e BRI1 interacts with AGB1 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:Myc-AGB1 , or pBRI1:BRI1-GFP ; 35S:Myc-AGB1 seedlings were incubated with GFP-Trap-A, respectively. Immunoprecipitates were examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. f BRI1 interacts with AGG3 in Arabidopsis. The extracted total proteins from pBRI1:BRI1-GFP ; 35S:GFP or pBRI1:BRI1-GFP ; 35S:Myc-AGG3 seedlings were incubated with GFP-Trap-A, and examined using anti-Myc and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. g BAK1 interacts with AGG3 in Arabidopsis. The extracted total proteins from 35S:GFP ; 35S:BAK1-HA and 35S:GFP-AGG3 ; 35S:BAK1-HA seedlings were incubated with GFP-Trap-A, and examined using anti-HA and anti-GFP antibodies, respectively. IP immunoprecipitation; IN input; IB immunoblot. Scale bars, 50 µm in c , d

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques: In Vitro, Western Blot, Fluorescence, Incubation, Immunoprecipitation

    BRI1 is required for the phosphorylation of AGB1 and AGG3. a BRI1 kinase domain (BRI1-KD) phosphorylates AGB1 in vitro, but bri1-301 kinase domain (bri1-301-KD) does not. The phosphorylated MBP-AGB1 (pMBP-AGB1), unphosphorylated (MBP-AGB1) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. b BRI1-KD phosphorylates AGG3 in vitro, but bri1-301-KD does not. The phosphorylated MBP-AGG3 (pMBP-AGG3), unphosphorylated (MBP-AGG3) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. c , d BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGB1 (L1) and 35S:GFP ; 35S:Myc-AGB1 (L2) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively c . Quantification of the phosphorylated Myc-AGB1 was relative to the immunoprecipitated Myc-AGB1 d . Values are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for L2 seedlings by Student’s t -test. e , f BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGG3 (L3) and 35S:GFP ; 35S:Myc-AGG3 (L4) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively ( e ). Quantification of the phosphorylated Myc-AGG3 was relative to the immunoprecipitated Myc-AGG3 f . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L4 seedlings by Student’s t -test. g , h BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from 35S:GFP-AGB1 (L5) and 35S:GFP-AGB1 ; bri1-301 (L6) seedlings were incubated with GFP-Trap-A, and detected using anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively g . Quantification of the phosphorylated GFP-AGB1 was relative to the immunoprecipitated GFP-AGB1 h . Values are given as mean ± SD, ( n = 3).** P < 0.01 compared with the value for L5 seedlings by Student’s t -test. i , j BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from 35S:GFP-AGG3 (L7) and 35S:GFP-AGG3 ; bri1-301 (L8) seedlings were incubated with GFP beads, and detected by immunoblot with anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively i . Quantification of the phosphorylated GFP-AGG3 was relative to the immunoprecipitated GFP-AGG3 j . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L7 seedlings by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 is required for the phosphorylation of AGB1 and AGG3. a BRI1 kinase domain (BRI1-KD) phosphorylates AGB1 in vitro, but bri1-301 kinase domain (bri1-301-KD) does not. The phosphorylated MBP-AGB1 (pMBP-AGB1), unphosphorylated (MBP-AGB1) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. b BRI1-KD phosphorylates AGG3 in vitro, but bri1-301-KD does not. The phosphorylated MBP-AGG3 (pMBP-AGG3), unphosphorylated (MBP-AGG3) and autophosphorylated GST-BRI1-KD (pGST-BRI1-KD) were separated by phos-tag SDS-PAGE. IN, input. c , d BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGB1 (L1) and 35S:GFP ; 35S:Myc-AGB1 (L2) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively c . Quantification of the phosphorylated Myc-AGB1 was relative to the immunoprecipitated Myc-AGB1 d . Values are given as mean ± SD ( n = 3). ** P < 0.01 compared with the value for L2 seedlings by Student’s t -test. e , f BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from pBRI1:BRI1-GFP ; 35S:Myc-AGG3 (L3) and 35S:GFP ; 35S:Myc-AGG3 (L4) seedlings were mixed with anti-Myc-Tag mouse mAb conjugated agarose beads and detected using anti-Myc and anti-phosphothreonine (pThr) antibodies, respectively ( e ). Quantification of the phosphorylated Myc-AGG3 was relative to the immunoprecipitated Myc-AGG3 f . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L4 seedlings by Student’s t -test. g , h BRI1 is required for the phosphorylation of AGB1 in Arabidopsis . Total proteins from 35S:GFP-AGB1 (L5) and 35S:GFP-AGB1 ; bri1-301 (L6) seedlings were incubated with GFP-Trap-A, and detected using anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively g . Quantification of the phosphorylated GFP-AGB1 was relative to the immunoprecipitated GFP-AGB1 h . Values are given as mean ± SD, ( n = 3).** P < 0.01 compared with the value for L5 seedlings by Student’s t -test. i , j BRI1 is required for the phosphorylation of AGG3 in Arabidopsis . Total proteins from 35S:GFP-AGG3 (L7) and 35S:GFP-AGG3 ; bri1-301 (L8) seedlings were incubated with GFP beads, and detected by immunoblot with anti-GFP and anti-phosphothreonine (pThr) antibodies, respectively i . Quantification of the phosphorylated GFP-AGG3 was relative to the immunoprecipitated GFP-AGG3 j . Values are given as mean ± SD ( n = 3).** P < 0.01 compared with the value for L7 seedlings by Student’s t -test

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques: In Vitro, SDS Page, Immunoprecipitation, Incubation, Western Blot

    BRI1 and BAK1 affect the interactions between GPA1 and AGB1. a BRI1 and BAK1 influences the interactions between GPA1 and AGB1 in Arabidopsis , respectively. The wild type, bri1-301 , and bak1-4 leaf protoplasts co-transformed with 35S:AGB1 - Myc and 35S : GPA1 - HA plasmids were grown in darkness for 14 h, and then treated without or with 2% glucose (G) for 5 h. Total proteins from these leaf protoplasts were incubated with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-HA antibodies, respectively. IN input; IP immunoprecipitation. b – d Quantification of relative interactions between GPA1 and AGB1 in wild type ( b ), bri1-301 ( c ) and bak1-4 ( d ) leaf protoplasts treated without or with 2% glucose as shown in a . The ratio was shown as immunoprecipitated GPA1-HA to input GPA1-HA. Values are given as mean ± SD ( n = 3) relative to the value for 0% glucose, set at 1. Values in b – d are given as mean ± SD, ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 affect the interactions between GPA1 and AGB1. a BRI1 and BAK1 influences the interactions between GPA1 and AGB1 in Arabidopsis , respectively. The wild type, bri1-301 , and bak1-4 leaf protoplasts co-transformed with 35S:AGB1 - Myc and 35S : GPA1 - HA plasmids were grown in darkness for 14 h, and then treated without or with 2% glucose (G) for 5 h. Total proteins from these leaf protoplasts were incubated with anti-Myc-Tag mouse mAb conjugated agarose beads, and detected using anti-Myc and anti-HA antibodies, respectively. IN input; IP immunoprecipitation. b – d Quantification of relative interactions between GPA1 and AGB1 in wild type ( b ), bri1-301 ( c ) and bak1-4 ( d ) leaf protoplasts treated without or with 2% glucose as shown in a . The ratio was shown as immunoprecipitated GPA1-HA to input GPA1-HA. Values are given as mean ± SD ( n = 3) relative to the value for 0% glucose, set at 1. Values in b – d are given as mean ± SD, ** P < 0.01 compared with the value for 0% glucose treatment by Student’s t -test

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques: Transformation Assay, Incubation, Immunoprecipitation

    BRI1 and BAK1 play key roles in sugar responses. a Different stages of dark-grown wild-type seedlings used for scoring sugar responses. b Comparison of developmental stages between Col-0, bri1-301 , and bak1-4 . Seedlings were grown vertically on medium supplemented with 1% glucose (G) or 1% mannitol (M) in the dark for 19 days ( n ≥ 83). c The percentage of Col-0, bri1-301 , and bak1-4 seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose (G) or 6% mannitol (M) under constant light condition for 10 days ( n ≥ 89). Values in b , c are given as mean ± SD. ** P < 0.01 compared with the wild type by Student’s t -test. Scale bars, 2 mm ( a )

    Journal: Nature Communications

    Article Title: BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis

    doi: 10.1038/s41467-018-03884-8

    Figure Lengend Snippet: BRI1 and BAK1 play key roles in sugar responses. a Different stages of dark-grown wild-type seedlings used for scoring sugar responses. b Comparison of developmental stages between Col-0, bri1-301 , and bak1-4 . Seedlings were grown vertically on medium supplemented with 1% glucose (G) or 1% mannitol (M) in the dark for 19 days ( n ≥ 83). c The percentage of Col-0, bri1-301 , and bak1-4 seedlings with green cotyledons. Seedlings were grown on medium supplemented with 6% glucose (G) or 6% mannitol (M) under constant light condition for 10 days ( n ≥ 89). Values in b , c are given as mean ± SD. ** P < 0.01 compared with the wild type by Student’s t -test. Scale bars, 2 mm ( a )

    Article Snippet: Immunoprecipitated BRI1 proteins were examined by anti-BRI1 (Abiocode R3283-4, 1/500) and anti-phosphoserine (Sigma P3430, 1/200) antibodies, respectively.

    Techniques: