antibody against maltose binding protein mbp  (New England Biolabs)


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    Anti MBP Magnetic Beads
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    Anti MBP Magnetic Beads 10 mg
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    New England Biolabs antibody against maltose binding protein mbp
    Anti MBP Magnetic Beads
    Anti MBP Magnetic Beads 10 mg
    https://www.bioz.com/result/antibody against maltose binding protein mbp/product/New England Biolabs
    Average 95 stars, based on 253 article reviews
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    antibody against maltose binding protein mbp - by Bioz Stars, 2020-10
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    Images

    1) Product Images from "Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens"

    Article Title: Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20170633

    ChiX D120A is inactive and incapable of rescuing ChiC secretion in a Δ chiX mutant. The pGEX-6P-1 chiX vector was modified to code for a D120A variant of ChiX, which was then isolated from E. coli BL21(DE3) following the protocol devised for the native protein. ( A ) Native ChiX protein (0.01 µM) was incubated with sacculi isolated from E. coli strain D456 and resultant peptidoglycan fragments were digested with cellosyl, reduced and separated by HPLC. No additional zinc was added in this experiment. Scale bar, 200 mAU. ( B ) As-purified ChiX D120A (1 µM) was incubated with sacculi. ( C ) The HPLC profile of sacculi isolated from E. coli strain D456 without exposure to ChiX. ( D ) Proposed structures of peptidoglycan fragments corresponding to numbered fractions in the profile shown in ( A–C ). ( E ) S. marcescens strain JJH05x (Δ chiX ) harbouring either pBAD18 chiX (‘Δ chiX , chiX ’) or pBAD18 chiX-D120A (‘Δ chiX , chiX D120A ’) was grown overnight in LB medium supplemented with 0.02% (w/v) l -arabinose. Cultures were then separated into whole cell (‘WC’) and culture supernatant (‘SN’) and analysed for ChiC secretion by SDS–PAGE and Western immunoblotting with anti-ChiC serum. A lysis control was also carried out by tracking the localisation of periplasmic MBP.
    Figure Legend Snippet: ChiX D120A is inactive and incapable of rescuing ChiC secretion in a Δ chiX mutant. The pGEX-6P-1 chiX vector was modified to code for a D120A variant of ChiX, which was then isolated from E. coli BL21(DE3) following the protocol devised for the native protein. ( A ) Native ChiX protein (0.01 µM) was incubated with sacculi isolated from E. coli strain D456 and resultant peptidoglycan fragments were digested with cellosyl, reduced and separated by HPLC. No additional zinc was added in this experiment. Scale bar, 200 mAU. ( B ) As-purified ChiX D120A (1 µM) was incubated with sacculi. ( C ) The HPLC profile of sacculi isolated from E. coli strain D456 without exposure to ChiX. ( D ) Proposed structures of peptidoglycan fragments corresponding to numbered fractions in the profile shown in ( A–C ). ( E ) S. marcescens strain JJH05x (Δ chiX ) harbouring either pBAD18 chiX (‘Δ chiX , chiX ’) or pBAD18 chiX-D120A (‘Δ chiX , chiX D120A ’) was grown overnight in LB medium supplemented with 0.02% (w/v) l -arabinose. Cultures were then separated into whole cell (‘WC’) and culture supernatant (‘SN’) and analysed for ChiC secretion by SDS–PAGE and Western immunoblotting with anti-ChiC serum. A lysis control was also carried out by tracking the localisation of periplasmic MBP.

    Techniques Used: Mutagenesis, Plasmid Preparation, Modification, Variant Assay, Isolation, Incubation, High Performance Liquid Chromatography, Purification, SDS Page, Western Blot, Lysis

    2) Product Images from "Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes ▿"

    Article Title: Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00716-09

    Cellular fractionation and localization of AmiC-GFP and AmiA-GFP. (A) W3110 (lanes 1 to 3) and BC202 (lanes 4 to 6) transformed with plasmid pTrcHis2-ELGFP6.1-TOPO were grown in LB medium with Amp without IPTG and converted to spheroplasts as described in Materials and Methods. The total cell lysate (T), spheroplast pellet (P), and spheroplast supernatant (S) were resolved by 12% SDS-PAGE and Western blotted with anti-GFP. (B and D) W3110 (lanes 1 to 3), BC202 (lanes 4 to 6), and BC202 harboring plasmid p- yghB (lanes 7 to 9) transformed with plasmid pTB28 were grown at 30°C in LB medium containing 0.1 mM IPTG and converted to spheroplasts. The total cell lysate, spheroplast pellet, and spheroplast supernatant were resolved by 12% SDS-PAGE and Western blotted with anti-GFP (B) or anti-MBP (D). (C) W3110 (lane 1), BC202 (lane 2), BC202 grown with 10 mM MgCl 2 (lane 3), BC202 harboring plasmid p- yghB (lane 4), and AD93 harboring plasmid pTB32 (lane 5) were grown at 30°C (or 37°C in the case of AD93) in LB medium containing 0.3 mM IPTG, and whole-cell lysates were analyzed by Western blotting using anti-GFP. Cleaved and uncleaved refer to signal peptidase-processed and -unprocessed forms of the protein that are expected to be found in the periplasm and cytoplasm, respectively. Longer exposures revealed no evidence of the processed protein in lanes 2 and 5 (not shown).
    Figure Legend Snippet: Cellular fractionation and localization of AmiC-GFP and AmiA-GFP. (A) W3110 (lanes 1 to 3) and BC202 (lanes 4 to 6) transformed with plasmid pTrcHis2-ELGFP6.1-TOPO were grown in LB medium with Amp without IPTG and converted to spheroplasts as described in Materials and Methods. The total cell lysate (T), spheroplast pellet (P), and spheroplast supernatant (S) were resolved by 12% SDS-PAGE and Western blotted with anti-GFP. (B and D) W3110 (lanes 1 to 3), BC202 (lanes 4 to 6), and BC202 harboring plasmid p- yghB (lanes 7 to 9) transformed with plasmid pTB28 were grown at 30°C in LB medium containing 0.1 mM IPTG and converted to spheroplasts. The total cell lysate, spheroplast pellet, and spheroplast supernatant were resolved by 12% SDS-PAGE and Western blotted with anti-GFP (B) or anti-MBP (D). (C) W3110 (lane 1), BC202 (lane 2), BC202 grown with 10 mM MgCl 2 (lane 3), BC202 harboring plasmid p- yghB (lane 4), and AD93 harboring plasmid pTB32 (lane 5) were grown at 30°C (or 37°C in the case of AD93) in LB medium containing 0.3 mM IPTG, and whole-cell lysates were analyzed by Western blotting using anti-GFP. Cleaved and uncleaved refer to signal peptidase-processed and -unprocessed forms of the protein that are expected to be found in the periplasm and cytoplasm, respectively. Longer exposures revealed no evidence of the processed protein in lanes 2 and 5 (not shown).

    Techniques Used: Cell Fractionation, Transformation Assay, Plasmid Preparation, SDS Page, Western Blot

    3) Product Images from "Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis"

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15603-3

    KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.
    Figure Legend Snippet: KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.

    Techniques Used: Luciferase, Activity Assay, Fluorescence, Construct, Negative Control, In Vitro, Incubation, Western Blot, Immunoprecipitation

    The KIX-PPD-MYC complex associates with the promoter of GIF1 and represses its expression. a The relative expression levels of GIF1 in the 0, 2 and 4 DAF (days after flowering) siliques of Col-0, kix8 - 1 kix9 - 1 , ppd1 - 2 ppd2 - cr and myc3 myc4 were detected by qPCR ( n = 3). Data was normalised with ACTIN2 . b The LUC activity of GIF1pro : LUC from the transient expression analysis in the Col-0 and myc3 myc4 protoplast ( n = 5). GIF1pro : LUC was cotransfected with different combinations of 35S : Myc - KIX8 (K8), 35S : Myc - KIX9 (K9), 35S : Myc - PPD1 (P1), 35S : Myc - PPD2 (P2), 35S : Myc - MYC3 (M3), 35 S : Myc - MYC4 (M4), and 35S : Myc - TPL (TPL) into the Col-0 and myc3 myc4 protoplast. The LUC and REN luciferase activities of GIF1pro : LUC were measured 40 h later after transfection. NC ( pGreen II_0800 - LUC ) was used as a negative control. c The schematic diagram of GIF1 promoter containing a typical G-box (5′-CACGTG-3′) sequence in F1 fragment. F1 - F4 represent DNA fragments used for ChIP-qPCR analysis. d ChIP-qPCR assays showing that KIX8/9 and PPD1/2 associate with the promoter of GIF1 by MYC3/4 in Arabidopsis ( n = 4). Chromatin from 1 to 4 DAF siliques of 35S : GFP , 35S : GFP - MYC3 ; myc3 , 35S : GFP - MYC4 ; myc4 , 35S:GFP-MYC3;myc3 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC4 ; myc4 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC3 ; myc3 kix8 - 1 kix9-1 , 35S : GFP - MYC4 ; myc4 kix8 - 1 kix9-1 , 35S : GFP - PPD1 ; ppd1 - 2 , 35S : GFP - PPD2 ; ppd2 - 1 , 35S : GFP - PPD1 ; ppd1 - 2 myc3 myc4 , 35S : GFP - PPD2 ; ppd2 - 1 myc3 myc4 , 35S : GFP - KIX8 ; kix8 - 1 , 35S : GFP - KIX9 ; kix9 - 1 , 35S : GFP - KIX8 ; kix8 - 1 myc3 myc4 and 35S : GFP - KIX9 ; kix9 - 1 myc3 myc4 were incubated with ChIP anti-GFP antibody and precipitated by ChIP protein A + G magnetic beads. The enrichment of fragments was determined by qPCR. The 35S : GFP plants acted as a control. The ACTIN7 promoter was used as a negative control. e The sequence of A and A - m probes for EMSA analysis. f , g The associations of MBP-MYC3 ( g ) and MBP-MYC4 ( i ) with the promoter of GIF1 were detected by EMSA. 5′-biotin- A / A - m probes were incubated with MBP or MBP-MYC3/4 and detected by ChIP western blot with the anti-biotin antibody. Error bars represent ±SE. Asterisk indicates significant difference, one-way ANOVA P -values: ** P
    Figure Legend Snippet: The KIX-PPD-MYC complex associates with the promoter of GIF1 and represses its expression. a The relative expression levels of GIF1 in the 0, 2 and 4 DAF (days after flowering) siliques of Col-0, kix8 - 1 kix9 - 1 , ppd1 - 2 ppd2 - cr and myc3 myc4 were detected by qPCR ( n = 3). Data was normalised with ACTIN2 . b The LUC activity of GIF1pro : LUC from the transient expression analysis in the Col-0 and myc3 myc4 protoplast ( n = 5). GIF1pro : LUC was cotransfected with different combinations of 35S : Myc - KIX8 (K8), 35S : Myc - KIX9 (K9), 35S : Myc - PPD1 (P1), 35S : Myc - PPD2 (P2), 35S : Myc - MYC3 (M3), 35 S : Myc - MYC4 (M4), and 35S : Myc - TPL (TPL) into the Col-0 and myc3 myc4 protoplast. The LUC and REN luciferase activities of GIF1pro : LUC were measured 40 h later after transfection. NC ( pGreen II_0800 - LUC ) was used as a negative control. c The schematic diagram of GIF1 promoter containing a typical G-box (5′-CACGTG-3′) sequence in F1 fragment. F1 - F4 represent DNA fragments used for ChIP-qPCR analysis. d ChIP-qPCR assays showing that KIX8/9 and PPD1/2 associate with the promoter of GIF1 by MYC3/4 in Arabidopsis ( n = 4). Chromatin from 1 to 4 DAF siliques of 35S : GFP , 35S : GFP - MYC3 ; myc3 , 35S : GFP - MYC4 ; myc4 , 35S:GFP-MYC3;myc3 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC4 ; myc4 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC3 ; myc3 kix8 - 1 kix9-1 , 35S : GFP - MYC4 ; myc4 kix8 - 1 kix9-1 , 35S : GFP - PPD1 ; ppd1 - 2 , 35S : GFP - PPD2 ; ppd2 - 1 , 35S : GFP - PPD1 ; ppd1 - 2 myc3 myc4 , 35S : GFP - PPD2 ; ppd2 - 1 myc3 myc4 , 35S : GFP - KIX8 ; kix8 - 1 , 35S : GFP - KIX9 ; kix9 - 1 , 35S : GFP - KIX8 ; kix8 - 1 myc3 myc4 and 35S : GFP - KIX9 ; kix9 - 1 myc3 myc4 were incubated with ChIP anti-GFP antibody and precipitated by ChIP protein A + G magnetic beads. The enrichment of fragments was determined by qPCR. The 35S : GFP plants acted as a control. The ACTIN7 promoter was used as a negative control. e The sequence of A and A - m probes for EMSA analysis. f , g The associations of MBP-MYC3 ( g ) and MBP-MYC4 ( i ) with the promoter of GIF1 were detected by EMSA. 5′-biotin- A / A - m probes were incubated with MBP or MBP-MYC3/4 and detected by ChIP western blot with the anti-biotin antibody. Error bars represent ±SE. Asterisk indicates significant difference, one-way ANOVA P -values: ** P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transfection, Negative Control, Sequencing, Chromatin Immunoprecipitation, Incubation, Magnetic Beads, Western Blot

    4) Product Images from "Apoptosis-dependent Externalization and Involvement in Apoptotic Cell Clearance of DmCaBP1, an Endoplasmic Reticulum Protein of Drosophila *"

    Article Title: Apoptosis-dependent Externalization and Involvement in Apoptotic Cell Clearance of DmCaBP1, an Endoplasmic Reticulum Protein of Drosophila *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.277921

    Surface DmCaBP1-mediated phagocytosis of latex beads and non-apoptotic cells. A , cycloheximide-treated S2 cells ( left panel ) and l(2)mbn cells ( right panel ) were incubated with recombinant DmCaBP1 proteins fused to GST and MBP, respectively. GST alone
    Figure Legend Snippet: Surface DmCaBP1-mediated phagocytosis of latex beads and non-apoptotic cells. A , cycloheximide-treated S2 cells ( left panel ) and l(2)mbn cells ( right panel ) were incubated with recombinant DmCaBP1 proteins fused to GST and MBP, respectively. GST alone

    Techniques Used: Incubation, Recombinant

    5) Product Images from "Proteome-scale purification of human proteins from bacteria"

    Article Title: Proteome-scale purification of human proteins from bacteria

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.042684199

    Test set proteins as purified with the GST tag ( A ) and the MBP tag ( B ). Bands of the correct size are indicated by a dot on the right side of the band. Fifteen percent of the total eluate was loaded on a 4–20% gradient SDS-gel and stained with GelCode Coomassie blue reagent.
    Figure Legend Snippet: Test set proteins as purified with the GST tag ( A ) and the MBP tag ( B ). Bands of the correct size are indicated by a dot on the right side of the band. Fifteen percent of the total eluate was loaded on a 4–20% gradient SDS-gel and stained with GelCode Coomassie blue reagent.

    Techniques Used: Purification, SDS-Gel, Staining

    GST- and MBP-tagged proteins are active. Autoradiograms of 32 P incorporation. ( A ) Equal amounts of bacterially purified GST- and MBP-tagged cyclin E were added to GST-cdk2 purified from insect cells in histone H1 kinase reactions. Both constructs activate cdk2 kinase activity. ( B ) p16 Ink4a specifically inhibits kinase activity. Equal volumes of 16 GST-tagged test set proteins were added to kinase reactions using cyclin D1/cdk4 purified from insect cells and C-terminal fragment of pRb as a substrate.
    Figure Legend Snippet: GST- and MBP-tagged proteins are active. Autoradiograms of 32 P incorporation. ( A ) Equal amounts of bacterially purified GST- and MBP-tagged cyclin E were added to GST-cdk2 purified from insect cells in histone H1 kinase reactions. Both constructs activate cdk2 kinase activity. ( B ) p16 Ink4a specifically inhibits kinase activity. Equal volumes of 16 GST-tagged test set proteins were added to kinase reactions using cyclin D1/cdk4 purified from insect cells and C-terminal fragment of pRb as a substrate.

    Techniques Used: Purification, Construct, Activity Assay

    6) Product Images from "A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria"

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    Journal: mBio

    doi: 10.1128/mBio.00809-18

    MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.
    Figure Legend Snippet: MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Techniques Used: SDS Page, Autoradiography, Activity Assay, Purification

    LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).
    Figure Legend Snippet: LovK and PhyK are phosphotransferases that are activated by MrrA~P. (A) Schematic representation of two possible modes of action of MrrA~P, phosphotransfer to and allosteric activation of PhyK and LovK. For reasons of simplicity, only PhyK is shown. (B) PhyK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, the kinase CC2874, MrrA, and different PhyK variants. Purified PhyK wild type or mutant variants harboring mutations in the G1 (G514A/G516A) or G2 (G526A) box of the ATP-binding site were used as indicated. The positions of phosphorylated proteins on the gel are indicated on the right. (C) LovK phosphorylation does not require a conserved CA domain. Phosphorylation reactions with radiolabeled ATP, kinase CC2874, MrrA, and different LovK variants. Purified LovK wild type or mutant variants harboring mutations in the G1 (G319A/G321A) or G2 (G332A) box ATP-binding site of the CA domain were used as indicated. MrrA dilution factors are indicated in each lane. The positions of phosphorylated proteins on the gel are indicated on the right. (D) Phosphotransfer to PhyR does not require a conserved CA domain. Phosphorylation reaction mixtures containing radiolabeled ATP, kinase CC2874, MrrA, PhyR, NepR, and different PhyK variants are as in panel C. The positions of phosphorylated proteins on the gel are indicated on the right. (E) Preparation of purified MrrA~P. Kinase CC2874 alone or with MrrA was phosphorylated with radiolabeled ATP (lanes 1 and 7), and CC2874 was subsequently removed using anti-MBP magnetic beads (lanes 2 and 8). Next, ATP was hydrolyzed by treating mixtures with hexokinase and glucose (lanes 3 and 9). CC2874 was added back to ATP-depleted samples, and mixtures were incubated for 0.5, 1.0, and 5 min (lanes 4 to 6 and 10 to 12). (F) Purified MrrA~P transfers phosphate to LovK. MrrA~P was prepared as in panel E (lane 1), CC2874 was removed (lane 2), and ATP was degraded (lane 3). Fresh CC2874 (lane 4) or LovK was added, and phosphotransfer from MrrA~P was monitored after 10 s, 20 s, 1 min, 2 min, 10 min, and 20 min (lanes 5 to 10, respectively). (G) ATP binding is not required for PhyK activity in the general stress response. Δ lovK strains harboring an empty vector (EV) or a plasmid expressing different phyK alleles from a cumate-inducible promoter were analyzed. Plasmid-driven variants of PhyK contained mutations in the G1 or G2 box of the ATP-binding pocket (see above) or in the conserved phosphoacceptor His371. SigT-dependent sigU promoter activity (Miller units) was determined using a lacZ promoter fusion in strains grown in the presence (+) or absence (−) of cumate. PhyK variants harbored a C-terminal 3×FLAG tag that allowed monitoring their expression by immunoblot analysis (lower panels). An immunoblot with anti-MreB antibodies is shown as a control. Note that the sigUp-lacZ reporter fusion used in these experiments differed from the one used in experiments above ( Fig. 1D ) and shows higher basal activity (compare wild-type PhyK and empty-vector control in panel G).

    Techniques Used: Activation Assay, Purification, Mutagenesis, Binding Assay, Magnetic Beads, Incubation, Activity Assay, Plasmid Preparation, Expressing

    7) Product Images from "The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201"

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01584

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P
    Figure Legend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay

    8) Product Images from "Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2"

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023668

    SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.
    Figure Legend Snippet: SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Construct, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.
    Figure Legend Snippet: SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Techniques Used: In Vitro, Immunoprecipitation, SDS Page, Staining

    9) Product Images from "The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201"

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01584

    ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P
    Figure Legend Snippet: ChIP-qPCR analyses showing the in vivo interaction between QslA/MvfR and the phz1 promoter region. (A) The ChIP fragment localized on the phz1 and pqsA promoters. (B) The 180-bp PCR products generated using immunoprecipitated DNA as templates and the primers specific to the phz1 promoter. Protein/DNA complexes isolated from the cell cultures were immunoprecipitated with the anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.). (C,D) qRT-PCR analysis of the relative enrichment of the phz1 and pqsA promoters in the immunoprecipitated DNA derived from the three strains. “+1” indicates the transcription initiation site and the arrows indicate the translational initiation site. Each bar represents the mean of three independent experiments; error bars indicate SDs. Statistical significance with respect to the strain Δ mvfR (MBP) is indicated by one, or two, or three asterisks ( P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, Polymerase Chain Reaction, Generated, Immunoprecipitation, Isolation, Magnetic Beads, Quantitative RT-PCR, Derivative Assay

    10) Product Images from "Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA"

    Article Title: Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2629

    Mapping protein and DNA components of the OCCM ( a - f ) 2D class averages and 3D reconstruction of OCCM with MBP fused to the C-terminus (CT) of Orc2 (Orc2-MBP) ( a ), the N-terminus (NT) of Mcm2 (MBP-Mcm2) ( b ), the NT of Mcm3 (MBP-Mcm3) ( c ), the CT of Mcm5 (MBP-Mcm5) ( d ), the NT of Cdt1 (MBP-Cdt1) ( e ), and the NT of Mcm6 (MBP-Mcm6) ( f ), respectively. In each left panel, the upper row shows two reference-free class averages, and the lower row shows the same images displayed at a higher contrast level (C=0.3). Each middle panel shows the surface rendered front, back, and bottom views of the 3D map of the MBP-fused OCCM complex. The peripheral MBP density is colored blue. The surface-rendering thresholds were lowered by ~20% to better visualize the small MBP density. Each right panel shows a vertical ( a-b ) or a horizontal section ( c-f ) of the 3D map of the MBP-fused OCCM (first column) in comparison to the corresponding section of that of the wild type OCCM (second column). The lower row is displayed at a higher contrast level than the upper row. The red arrows point to the MBP density at the peripheral of OCCM. All MBP fusion complexes were imaged by cryo-EM exception for MBP-Mcm3 ( c ) that was by negative stained EM. All fusion complexes were cleaved off the plasmid DNA by DNases I except for MBP-Mcm6 that was by Alu I ( f ). ( g ) Reference-free class averages of wtOCCM with their plasmid DNA digested either by DNase I (upper row) or by Alu I (lower row). Blue arrows point to dsDNA stub on the top ORC-Cdc6 region of OCCM. ( h ) MCM2-7 organization as mapped by the four MBP-fused Mcm subunits (red), viewed from the N-terminal end of MCM2-7. Box size is 37 nm in the left panels, 34 nm in the right panels in ( a-f ), and 31 nm in ( g ). 3D maps are on same scale.
    Figure Legend Snippet: Mapping protein and DNA components of the OCCM ( a - f ) 2D class averages and 3D reconstruction of OCCM with MBP fused to the C-terminus (CT) of Orc2 (Orc2-MBP) ( a ), the N-terminus (NT) of Mcm2 (MBP-Mcm2) ( b ), the NT of Mcm3 (MBP-Mcm3) ( c ), the CT of Mcm5 (MBP-Mcm5) ( d ), the NT of Cdt1 (MBP-Cdt1) ( e ), and the NT of Mcm6 (MBP-Mcm6) ( f ), respectively. In each left panel, the upper row shows two reference-free class averages, and the lower row shows the same images displayed at a higher contrast level (C=0.3). Each middle panel shows the surface rendered front, back, and bottom views of the 3D map of the MBP-fused OCCM complex. The peripheral MBP density is colored blue. The surface-rendering thresholds were lowered by ~20% to better visualize the small MBP density. Each right panel shows a vertical ( a-b ) or a horizontal section ( c-f ) of the 3D map of the MBP-fused OCCM (first column) in comparison to the corresponding section of that of the wild type OCCM (second column). The lower row is displayed at a higher contrast level than the upper row. The red arrows point to the MBP density at the peripheral of OCCM. All MBP fusion complexes were imaged by cryo-EM exception for MBP-Mcm3 ( c ) that was by negative stained EM. All fusion complexes were cleaved off the plasmid DNA by DNases I except for MBP-Mcm6 that was by Alu I ( f ). ( g ) Reference-free class averages of wtOCCM with their plasmid DNA digested either by DNase I (upper row) or by Alu I (lower row). Blue arrows point to dsDNA stub on the top ORC-Cdc6 region of OCCM. ( h ) MCM2-7 organization as mapped by the four MBP-fused Mcm subunits (red), viewed from the N-terminal end of MCM2-7. Box size is 37 nm in the left panels, 34 nm in the right panels in ( a-f ), and 31 nm in ( g ). 3D maps are on same scale.

    Techniques Used: Staining, Plasmid Preparation

    In vitro assembly of the OCCM complex ( a ) Model for Cdc6 recruitment to the replication origin readies the ORC for loading of MCM2-7. ( b ) Averaged cryo-EM images of the in vitro assembled OCCM. For scale, the box size is 27 nm. An enlarged view with the top area tentatively assigned to ORC-Cdc6 and the lower region to Cdt1-MCM2-7. ( c ) Mcm2 IP identifies the OCCM components. Using purified ORC, Cdc6, Cdt1, MCM2-7 (lanes 1-4) and origin DNA OCCM was assembled in the presence of ATPγS. OCCM was cleaved off from the plasmid DNA with DNase I and immunoprecipitated with an anti-Mcm2 antibody (lanes 5-7) or with anti-MBP control antibody (lanes 8-10). Asterisk marks nonspecific proteins from antibody-conjugated beads.
    Figure Legend Snippet: In vitro assembly of the OCCM complex ( a ) Model for Cdc6 recruitment to the replication origin readies the ORC for loading of MCM2-7. ( b ) Averaged cryo-EM images of the in vitro assembled OCCM. For scale, the box size is 27 nm. An enlarged view with the top area tentatively assigned to ORC-Cdc6 and the lower region to Cdt1-MCM2-7. ( c ) Mcm2 IP identifies the OCCM components. Using purified ORC, Cdc6, Cdt1, MCM2-7 (lanes 1-4) and origin DNA OCCM was assembled in the presence of ATPγS. OCCM was cleaved off from the plasmid DNA with DNase I and immunoprecipitated with an anti-Mcm2 antibody (lanes 5-7) or with anti-MBP control antibody (lanes 8-10). Asterisk marks nonspecific proteins from antibody-conjugated beads.

    Techniques Used: In Vitro, Purification, Plasmid Preparation, Immunoprecipitation

    11) Product Images from "A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria"

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria

    Journal: mBio

    doi: 10.1128/mBio.00809-18

    MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.
    Figure Legend Snippet: MrrA phosphorylates components of the general stress response. (A) MrrA transfers phosphate to LovK and PhyR. Five hundred micromoles ATP and 2.5 µCi [γ- 32 P]ATP (3,000 Ci mmol −1 ) were mixed with the proteins indicated, reactions were carried out for 15 min at room temperature, and reaction mixtures were analyzed by SDS-PAGE and autoradiography. Note that LovK does not display autokinase activity by itself but is readily phosphorylated if MrrA is present. The positions of phosphorylated proteins on the gels are indicated on the right. (B) MrrA transfers phosphate to PhyK and PhyR. Phosphorylation reactions were assembled and run as in panel A. The results obtained for PhyK were similar to the results obtained for LovK. PhyK does not display autokinase activity but is readily phosphorylated if MrrA is present. Note that the MBP-CC2874 preparation contains a fraction of cleaved protein that represents CC2874 without MBP tag, such that autophosphorylation of (MBP-)CC2874 yields two radiolabeled bands (lane 3), the lower one of which migrates only slightly faster than the PhyK band. The positions of phosphorylated proteins on the gels are indicated on the right. (C) Phosphorylation of MrrA and LovK is rapid, while phosphorylation of PhyR is slow. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, PhyR, and NepR were preincubated for 30 min (premix). Purified MrrA was then added to the reaction mixtures, and samples were taken at the time points indicated. The positions of phosphorylated proteins on the gels are indicated on the right with proteins present in the premix being highlighted in blue (C to E). (D) PhyR phosphorylation through LovK is slow and inefficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, LovK, MrrA, and NepR were preincubated for 30 min (premix). Purified PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated. (E) PhyR phosphorylation through PhyK is rapid and efficient. Phosphorylation reaction mixtures with radiolabeled ATP and purified CC2874, PhyK, MrrA, and NepR were preincubated for 30 min (premix). PhyR was then added to the reaction mixtures, and samples were taken at the time points indicated.

    Techniques Used: SDS Page, Autoradiography, Activity Assay, Purification

    12) Product Images from "Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2"

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0023668

    SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.
    Figure Legend Snippet: SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles. (C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these (Lanes 1–4 respectively) at 55°C for 1 hour in 1× ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using a phosphoimager. Fragment sizes are indicated.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing, Construct, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.
    Figure Legend Snippet: SIRV2 Hjr interacts with SIRV2gp26 coat protein in vitro . The interaction of MBP-Hjr and SIRV2gp26 coat protein was confirmed by immunoprecipitation. Protein complexes were eluted and analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1: SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4: anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-MBP beads:MBP-Hjr+SIRV2gp26.

    Techniques Used: In Vitro, Immunoprecipitation, SDS Page, Staining

    13) Product Images from "Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis"

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15603-3

    KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.
    Figure Legend Snippet: KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.

    Techniques Used: Luciferase, Activity Assay, Fluorescence, Construct, Negative Control, In Vitro, Incubation, Western Blot, Immunoprecipitation

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    Article Snippet: .. Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). .. Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour.

    Magnetic Beads:

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet. .. Hexokinase and glucose were added to the supernatant as described above and incubated for 10 min to deplete remaining ATP.

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples. .. After incubation at 4°C overnight, beads were pelleted and washed three times with 0.1 M NaPhosphate buffer (pH 8.0).

    Article Title: Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2
    Article Snippet: .. Interaction between Hjr and SIRV2gp26 coat protein To directly test the interaction between Hjr and SIRV2gp26 coat protein in vitro , anti-MBP::MBP-Hjr magnetic beads (hereafter called MBP-Hjr affinity beads) were prepared by mixing 10 µg MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated in NEBuffer 3 (New England Biolabs). .. Bead complexes were mixed thoroughly and incubated at 4°C with shaking for 1 hour.

    Purification:

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis
    Article Snippet: .. The biotin-labelled and unlabelled probes were synthesised and incubated with MBP, MBP-MYC3, or MBP-MYC4 at room temperature for 20 min. Proteins were purified with anti-MBP agarose beads (NEB, E8037s). .. The interactions were detected with an anti-biotin antibody (1:3000, Invitrogen, 03-3720).

    Immunoprecipitation:

    Article Title: The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201
    Article Snippet: .. The immunoprecipitation reaction was initiated by the addition of anti-MBP magnetic beads (Cat # E8037S; New England Biolabs, Inc.), to each of the remaining samples. .. After incubation at 4°C overnight, beads were pelleted and washed three times with 0.1 M NaPhosphate buffer (pH 8.0).

    Incubation:

    Article Title: A Single-Domain Response Regulator Functions as an Integrating Hub To Coordinate General Stress Response and Development in Alphaproteobacteria
    Article Snippet: .. CC2874 (0.2 µM) and MrrA (100 µM) were prephosphorylated for 1 h. Twenty-five microliters of anti-MBP magnetic beads (New England Biolabs; E8037S) was added and incubated for 1 h. Beads were then concentrated using a magnet. .. Hexokinase and glucose were added to the supernatant as described above and incubated for 10 min to deplete remaining ATP.

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis
    Article Snippet: .. The biotin-labelled and unlabelled probes were synthesised and incubated with MBP, MBP-MYC3, or MBP-MYC4 at room temperature for 20 min. Proteins were purified with anti-MBP agarose beads (NEB, E8037s). .. The interactions were detected with an anti-biotin antibody (1:3000, Invitrogen, 03-3720).

    Western Blot:

    Article Title: Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes ▿
    Article Snippet: .. Western blotting was carried out as described previously , with primary antibody anti-GFP JL-8 (Clontech Laboratories) or anti-maltose binding protein (MBP) (New England Biolabs) used at a dilution of 1:10,000. .. The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Scientific), and detection was performed with an Immun-Star kit (Bio-Rad Laboratories).

    Article Title: Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens
    Article Snippet: .. These samples were then separated by SDS–PAGE and analysed by Western immunoblotting using antisera for ChiC [ ] and a commercial antibody against maltose-binding protein (MBP) (New England Biolabs). .. The crystal structure of S. marcescens ChiX The S. marcescens ChiX protein was overproduced as a GST fusion in E. coli before being isolated, separated from GST by proteolysis and then finally subjected to size-exclusion chromatography (Supplementary Figure S1).

    Binding Assay:

    Article Title: Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes ▿
    Article Snippet: .. Western blotting was carried out as described previously , with primary antibody anti-GFP JL-8 (Clontech Laboratories) or anti-maltose binding protein (MBP) (New England Biolabs) used at a dilution of 1:10,000. .. The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG (Thermo Scientific), and detection was performed with an Immun-Star kit (Bio-Rad Laboratories).

    SDS Page:

    Article Title: Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens
    Article Snippet: .. These samples were then separated by SDS–PAGE and analysed by Western immunoblotting using antisera for ChiC [ ] and a commercial antibody against maltose-binding protein (MBP) (New England Biolabs). .. The crystal structure of S. marcescens ChiX The S. marcescens ChiX protein was overproduced as a GST fusion in E. coli before being isolated, separated from GST by proteolysis and then finally subjected to size-exclusion chromatography (Supplementary Figure S1).

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    New England Biolabs antibody against maltose binding protein mbp
    ChiX D120A is inactive and incapable of rescuing ChiC secretion in a Δ chiX mutant. The pGEX-6P-1 chiX vector was modified to code for a D120A variant of ChiX, which was then isolated from E. coli BL21(DE3) following the protocol devised for the native protein. ( A ) Native ChiX protein (0.01 µM) was incubated with sacculi isolated from E. coli strain D456 and resultant peptidoglycan fragments were digested with cellosyl, reduced and separated by HPLC. No additional zinc was added in this experiment. Scale bar, 200 mAU. ( B ) As-purified ChiX D120A (1 µM) was incubated with sacculi. ( C ) The HPLC profile of sacculi isolated from E. coli strain D456 without exposure to ChiX. ( D ) Proposed structures of peptidoglycan fragments corresponding to numbered fractions in the profile shown in ( A–C ). ( E ) S. marcescens strain JJH05x (Δ chiX ) harbouring either pBAD18 chiX (‘Δ chiX , chiX ’) or pBAD18 chiX-D120A (‘Δ chiX , chiX D120A ’) was grown overnight in LB medium supplemented with 0.02% (w/v) l -arabinose. Cultures were then separated into whole cell (‘WC’) and culture supernatant (‘SN’) and analysed for ChiC secretion by SDS–PAGE and Western <t>immunoblotting</t> with anti-ChiC serum. A lysis control was also carried out by tracking the localisation of periplasmic <t>MBP.</t>
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    ChiX D120A is inactive and incapable of rescuing ChiC secretion in a Δ chiX mutant. The pGEX-6P-1 chiX vector was modified to code for a D120A variant of ChiX, which was then isolated from E. coli BL21(DE3) following the protocol devised for the native protein. ( A ) Native ChiX protein (0.01 µM) was incubated with sacculi isolated from E. coli strain D456 and resultant peptidoglycan fragments were digested with cellosyl, reduced and separated by HPLC. No additional zinc was added in this experiment. Scale bar, 200 mAU. ( B ) As-purified ChiX D120A (1 µM) was incubated with sacculi. ( C ) The HPLC profile of sacculi isolated from E. coli strain D456 without exposure to ChiX. ( D ) Proposed structures of peptidoglycan fragments corresponding to numbered fractions in the profile shown in ( A–C ). ( E ) S. marcescens strain JJH05x (Δ chiX ) harbouring either pBAD18 chiX (‘Δ chiX , chiX ’) or pBAD18 chiX-D120A (‘Δ chiX , chiX D120A ’) was grown overnight in LB medium supplemented with 0.02% (w/v) l -arabinose. Cultures were then separated into whole cell (‘WC’) and culture supernatant (‘SN’) and analysed for ChiC secretion by SDS–PAGE and Western immunoblotting with anti-ChiC serum. A lysis control was also carried out by tracking the localisation of periplasmic MBP.

    Journal: Biochemical Journal

    Article Title: Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens

    doi: 10.1042/BCJ20170633

    Figure Lengend Snippet: ChiX D120A is inactive and incapable of rescuing ChiC secretion in a Δ chiX mutant. The pGEX-6P-1 chiX vector was modified to code for a D120A variant of ChiX, which was then isolated from E. coli BL21(DE3) following the protocol devised for the native protein. ( A ) Native ChiX protein (0.01 µM) was incubated with sacculi isolated from E. coli strain D456 and resultant peptidoglycan fragments were digested with cellosyl, reduced and separated by HPLC. No additional zinc was added in this experiment. Scale bar, 200 mAU. ( B ) As-purified ChiX D120A (1 µM) was incubated with sacculi. ( C ) The HPLC profile of sacculi isolated from E. coli strain D456 without exposure to ChiX. ( D ) Proposed structures of peptidoglycan fragments corresponding to numbered fractions in the profile shown in ( A–C ). ( E ) S. marcescens strain JJH05x (Δ chiX ) harbouring either pBAD18 chiX (‘Δ chiX , chiX ’) or pBAD18 chiX-D120A (‘Δ chiX , chiX D120A ’) was grown overnight in LB medium supplemented with 0.02% (w/v) l -arabinose. Cultures were then separated into whole cell (‘WC’) and culture supernatant (‘SN’) and analysed for ChiC secretion by SDS–PAGE and Western immunoblotting with anti-ChiC serum. A lysis control was also carried out by tracking the localisation of periplasmic MBP.

    Article Snippet: These samples were then separated by SDS–PAGE and analysed by Western immunoblotting using antisera for ChiC [ ] and a commercial antibody against maltose-binding protein (MBP) (New England Biolabs).

    Techniques: Mutagenesis, Plasmid Preparation, Modification, Variant Assay, Isolation, Incubation, High Performance Liquid Chromatography, Purification, SDS Page, Western Blot, Lysis

    Cellular fractionation and localization of AmiC-GFP and AmiA-GFP. (A) W3110 (lanes 1 to 3) and BC202 (lanes 4 to 6) transformed with plasmid pTrcHis2-ELGFP6.1-TOPO were grown in LB medium with Amp without IPTG and converted to spheroplasts as described in Materials and Methods. The total cell lysate (T), spheroplast pellet (P), and spheroplast supernatant (S) were resolved by 12% SDS-PAGE and Western blotted with anti-GFP. (B and D) W3110 (lanes 1 to 3), BC202 (lanes 4 to 6), and BC202 harboring plasmid p- yghB (lanes 7 to 9) transformed with plasmid pTB28 were grown at 30°C in LB medium containing 0.1 mM IPTG and converted to spheroplasts. The total cell lysate, spheroplast pellet, and spheroplast supernatant were resolved by 12% SDS-PAGE and Western blotted with anti-GFP (B) or anti-MBP (D). (C) W3110 (lane 1), BC202 (lane 2), BC202 grown with 10 mM MgCl 2 (lane 3), BC202 harboring plasmid p- yghB (lane 4), and AD93 harboring plasmid pTB32 (lane 5) were grown at 30°C (or 37°C in the case of AD93) in LB medium containing 0.3 mM IPTG, and whole-cell lysates were analyzed by Western blotting using anti-GFP. Cleaved and uncleaved refer to signal peptidase-processed and -unprocessed forms of the protein that are expected to be found in the periplasm and cytoplasm, respectively. Longer exposures revealed no evidence of the processed protein in lanes 2 and 5 (not shown).

    Journal: Journal of Bacteriology

    Article Title: Inefficient Tat-Dependent Export of Periplasmic Amidases in an Escherichia coli Strain with Mutations in Two DedA Family Genes ▿

    doi: 10.1128/JB.00716-09

    Figure Lengend Snippet: Cellular fractionation and localization of AmiC-GFP and AmiA-GFP. (A) W3110 (lanes 1 to 3) and BC202 (lanes 4 to 6) transformed with plasmid pTrcHis2-ELGFP6.1-TOPO were grown in LB medium with Amp without IPTG and converted to spheroplasts as described in Materials and Methods. The total cell lysate (T), spheroplast pellet (P), and spheroplast supernatant (S) were resolved by 12% SDS-PAGE and Western blotted with anti-GFP. (B and D) W3110 (lanes 1 to 3), BC202 (lanes 4 to 6), and BC202 harboring plasmid p- yghB (lanes 7 to 9) transformed with plasmid pTB28 were grown at 30°C in LB medium containing 0.1 mM IPTG and converted to spheroplasts. The total cell lysate, spheroplast pellet, and spheroplast supernatant were resolved by 12% SDS-PAGE and Western blotted with anti-GFP (B) or anti-MBP (D). (C) W3110 (lane 1), BC202 (lane 2), BC202 grown with 10 mM MgCl 2 (lane 3), BC202 harboring plasmid p- yghB (lane 4), and AD93 harboring plasmid pTB32 (lane 5) were grown at 30°C (or 37°C in the case of AD93) in LB medium containing 0.3 mM IPTG, and whole-cell lysates were analyzed by Western blotting using anti-GFP. Cleaved and uncleaved refer to signal peptidase-processed and -unprocessed forms of the protein that are expected to be found in the periplasm and cytoplasm, respectively. Longer exposures revealed no evidence of the processed protein in lanes 2 and 5 (not shown).

    Article Snippet: Western blotting was carried out as described previously , with primary antibody anti-GFP JL-8 (Clontech Laboratories) or anti-maltose binding protein (MBP) (New England Biolabs) used at a dilution of 1:10,000.

    Techniques: Cell Fractionation, Transformation Assay, Plasmid Preparation, SDS Page, Western Blot

    KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.

    Journal: Nature Communications

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis

    doi: 10.1038/s41467-020-15603-3

    Figure Lengend Snippet: KIX8/9, PPD1/2, and MYC3/4 form a complex in Arabidopsis. a Split luciferase complementation assays showing the interactions between PPD1/2 and MYC3/4. MYC3/4-nLUC and cLUC-PPD1/2 were coexpressed in N. benthamiana leaves. The luciferase activity was detected at 2 days later after infiltration. b The FRET-FLIM assays showing that MYC3/4 interact with PPD1/2 in N. benthamiana leaves. CFP fluorescence lifetime was obtained at 2 days later after coinfiltrating with different combinations of 35S : MYC3 - CFP , 35S : MYC4 - CFP , 35S : PPD1 - YFP , 35S : PPD2 - YFP , and 35S : DEL1 - YFP constructs. 35S : DEL1 - YFP was used as a negative control. c Pull-down analyses showing the interactions between PPD1/2 and MYC3/4 in vitro. GST-MYC3 and GST-MYC4 were incubated with MBP-PPD1, MBP-PPD2, and MBP, respectively. Proteins were pulled down by MBP-Trap-A agarose beads and detected by Western blot with anti-GST or anti-MBP antibody. d Co-immunoprecipitation analyses showing the interactions between PPD1/2 and MYC3/4 in Arabidopsis. Total protein extracts of 35S : Myc - PPD1 / 2 ; 35S : GFP and 35S : Myc - PPD1 / 2 ; 35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody. e Pull-down analyses showing that PPD1/2 are required for the interactions between KIX8 and MYC3/4. His-KIX8 was incubated with GST-MYC3 or GST-MYC4 and MBP-PPD1 or MBP-PPD2. Proteins were pulled down by the Ni-NTA agarose beads and detected by Western blot with anti-GST, anti-MBP, or anti-His antibody. f Co-immunoprecipitation analyses showing that KIX8/9 and MYC3/4 are in a protein complex in Arabidopsis. Total protein extracts of 35S : Myc - KIX8 / 9;35S : GFP and 35S : Myc - KIX8 / 9;35S : GFP - MYC3 / 4 plants were incubated with GFP-Trap agarose beads. Precipitates were detected by Western blot with anti-GFP or anti-Myc antibody.

    Article Snippet: The biotin-labelled and unlabelled probes were synthesised and incubated with MBP, MBP-MYC3, or MBP-MYC4 at room temperature for 20 min. Proteins were purified with anti-MBP agarose beads (NEB, E8037s).

    Techniques: Luciferase, Activity Assay, Fluorescence, Construct, Negative Control, In Vitro, Incubation, Western Blot, Immunoprecipitation

    The KIX-PPD-MYC complex associates with the promoter of GIF1 and represses its expression. a The relative expression levels of GIF1 in the 0, 2 and 4 DAF (days after flowering) siliques of Col-0, kix8 - 1 kix9 - 1 , ppd1 - 2 ppd2 - cr and myc3 myc4 were detected by qPCR ( n = 3). Data was normalised with ACTIN2 . b The LUC activity of GIF1pro : LUC from the transient expression analysis in the Col-0 and myc3 myc4 protoplast ( n = 5). GIF1pro : LUC was cotransfected with different combinations of 35S : Myc - KIX8 (K8), 35S : Myc - KIX9 (K9), 35S : Myc - PPD1 (P1), 35S : Myc - PPD2 (P2), 35S : Myc - MYC3 (M3), 35 S : Myc - MYC4 (M4), and 35S : Myc - TPL (TPL) into the Col-0 and myc3 myc4 protoplast. The LUC and REN luciferase activities of GIF1pro : LUC were measured 40 h later after transfection. NC ( pGreen II_0800 - LUC ) was used as a negative control. c The schematic diagram of GIF1 promoter containing a typical G-box (5′-CACGTG-3′) sequence in F1 fragment. F1 - F4 represent DNA fragments used for ChIP-qPCR analysis. d ChIP-qPCR assays showing that KIX8/9 and PPD1/2 associate with the promoter of GIF1 by MYC3/4 in Arabidopsis ( n = 4). Chromatin from 1 to 4 DAF siliques of 35S : GFP , 35S : GFP - MYC3 ; myc3 , 35S : GFP - MYC4 ; myc4 , 35S:GFP-MYC3;myc3 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC4 ; myc4 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC3 ; myc3 kix8 - 1 kix9-1 , 35S : GFP - MYC4 ; myc4 kix8 - 1 kix9-1 , 35S : GFP - PPD1 ; ppd1 - 2 , 35S : GFP - PPD2 ; ppd2 - 1 , 35S : GFP - PPD1 ; ppd1 - 2 myc3 myc4 , 35S : GFP - PPD2 ; ppd2 - 1 myc3 myc4 , 35S : GFP - KIX8 ; kix8 - 1 , 35S : GFP - KIX9 ; kix9 - 1 , 35S : GFP - KIX8 ; kix8 - 1 myc3 myc4 and 35S : GFP - KIX9 ; kix9 - 1 myc3 myc4 were incubated with ChIP anti-GFP antibody and precipitated by ChIP protein A + G magnetic beads. The enrichment of fragments was determined by qPCR. The 35S : GFP plants acted as a control. The ACTIN7 promoter was used as a negative control. e The sequence of A and A - m probes for EMSA analysis. f , g The associations of MBP-MYC3 ( g ) and MBP-MYC4 ( i ) with the promoter of GIF1 were detected by EMSA. 5′-biotin- A / A - m probes were incubated with MBP or MBP-MYC3/4 and detected by ChIP western blot with the anti-biotin antibody. Error bars represent ±SE. Asterisk indicates significant difference, one-way ANOVA P -values: ** P

    Journal: Nature Communications

    Article Title: Transcriptional repression of GIF1 by the KIX-PPD-MYC repressor complex controls seed size in Arabidopsis

    doi: 10.1038/s41467-020-15603-3

    Figure Lengend Snippet: The KIX-PPD-MYC complex associates with the promoter of GIF1 and represses its expression. a The relative expression levels of GIF1 in the 0, 2 and 4 DAF (days after flowering) siliques of Col-0, kix8 - 1 kix9 - 1 , ppd1 - 2 ppd2 - cr and myc3 myc4 were detected by qPCR ( n = 3). Data was normalised with ACTIN2 . b The LUC activity of GIF1pro : LUC from the transient expression analysis in the Col-0 and myc3 myc4 protoplast ( n = 5). GIF1pro : LUC was cotransfected with different combinations of 35S : Myc - KIX8 (K8), 35S : Myc - KIX9 (K9), 35S : Myc - PPD1 (P1), 35S : Myc - PPD2 (P2), 35S : Myc - MYC3 (M3), 35 S : Myc - MYC4 (M4), and 35S : Myc - TPL (TPL) into the Col-0 and myc3 myc4 protoplast. The LUC and REN luciferase activities of GIF1pro : LUC were measured 40 h later after transfection. NC ( pGreen II_0800 - LUC ) was used as a negative control. c The schematic diagram of GIF1 promoter containing a typical G-box (5′-CACGTG-3′) sequence in F1 fragment. F1 - F4 represent DNA fragments used for ChIP-qPCR analysis. d ChIP-qPCR assays showing that KIX8/9 and PPD1/2 associate with the promoter of GIF1 by MYC3/4 in Arabidopsis ( n = 4). Chromatin from 1 to 4 DAF siliques of 35S : GFP , 35S : GFP - MYC3 ; myc3 , 35S : GFP - MYC4 ; myc4 , 35S:GFP-MYC3;myc3 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC4 ; myc4 ppd1 - 2 ppd2 - cr , 35S : GFP - MYC3 ; myc3 kix8 - 1 kix9-1 , 35S : GFP - MYC4 ; myc4 kix8 - 1 kix9-1 , 35S : GFP - PPD1 ; ppd1 - 2 , 35S : GFP - PPD2 ; ppd2 - 1 , 35S : GFP - PPD1 ; ppd1 - 2 myc3 myc4 , 35S : GFP - PPD2 ; ppd2 - 1 myc3 myc4 , 35S : GFP - KIX8 ; kix8 - 1 , 35S : GFP - KIX9 ; kix9 - 1 , 35S : GFP - KIX8 ; kix8 - 1 myc3 myc4 and 35S : GFP - KIX9 ; kix9 - 1 myc3 myc4 were incubated with ChIP anti-GFP antibody and precipitated by ChIP protein A + G magnetic beads. The enrichment of fragments was determined by qPCR. The 35S : GFP plants acted as a control. The ACTIN7 promoter was used as a negative control. e The sequence of A and A - m probes for EMSA analysis. f , g The associations of MBP-MYC3 ( g ) and MBP-MYC4 ( i ) with the promoter of GIF1 were detected by EMSA. 5′-biotin- A / A - m probes were incubated with MBP or MBP-MYC3/4 and detected by ChIP western blot with the anti-biotin antibody. Error bars represent ±SE. Asterisk indicates significant difference, one-way ANOVA P -values: ** P

    Article Snippet: The biotin-labelled and unlabelled probes were synthesised and incubated with MBP, MBP-MYC3, or MBP-MYC4 at room temperature for 20 min. Proteins were purified with anti-MBP agarose beads (NEB, E8037s).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transfection, Negative Control, Sequencing, Chromatin Immunoprecipitation, Incubation, Magnetic Beads, Western Blot

    Surface DmCaBP1-mediated phagocytosis of latex beads and non-apoptotic cells. A , cycloheximide-treated S2 cells ( left panel ) and l(2)mbn cells ( right panel ) were incubated with recombinant DmCaBP1 proteins fused to GST and MBP, respectively. GST alone

    Journal: The Journal of Biological Chemistry

    Article Title: Apoptosis-dependent Externalization and Involvement in Apoptotic Cell Clearance of DmCaBP1, an Endoplasmic Reticulum Protein of Drosophila *

    doi: 10.1074/jbc.M111.277921

    Figure Lengend Snippet: Surface DmCaBP1-mediated phagocytosis of latex beads and non-apoptotic cells. A , cycloheximide-treated S2 cells ( left panel ) and l(2)mbn cells ( right panel ) were incubated with recombinant DmCaBP1 proteins fused to GST and MBP, respectively. GST alone

    Article Snippet: Anti-GST mAb was purchased from Millipore, and anti-maltose-binding protein (MBP) antibody was from New England Biolabs.

    Techniques: Incubation, Recombinant