anti mbp monoclonal antibody  (New England Biolabs)


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    Structured Review

    New England Biolabs anti mbp monoclonal antibody
    Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in <t>V5-tagged</t> Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding <t>protein-(MS2-MBP)-conjugated</t> agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.
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    Images

    1) Product Images from "Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling"

    Article Title: Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling

    Journal: Nature Communications

    doi: 10.1038/s41467-021-23459-4

    Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in V5-tagged Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding protein-(MS2-MBP)-conjugated agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.
    Figure Legend Snippet: Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in V5-tagged Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding protein-(MS2-MBP)-conjugated agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.

    Techniques Used: Immunoprecipitation, Binding Assay, Irradiation

    2) Product Images from "A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4"

    Article Title: A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4

    Journal: Journal of Virology

    doi:

    SDS-PAGE of MBP-VP4 fusion proteins. The samples of MBP fusion protein containing either wild-type or mutant forms of the leucine zipper region of VP4 were analyzed by SDS-PAGE (10% polyacrylamide). Lanes: 1, purified MBP; 2, purified fusion protein derived from plasmid MBP/VP4LZ expressing the wild-type leucine zipper region; 3 and 4, standard molecular mass markers; 5, purified fusion protein derived from plasmid MBP/VP4LZP expressing the mutant (L537P) leucine zipper region.
    Figure Legend Snippet: SDS-PAGE of MBP-VP4 fusion proteins. The samples of MBP fusion protein containing either wild-type or mutant forms of the leucine zipper region of VP4 were analyzed by SDS-PAGE (10% polyacrylamide). Lanes: 1, purified MBP; 2, purified fusion protein derived from plasmid MBP/VP4LZ expressing the wild-type leucine zipper region; 3 and 4, standard molecular mass markers; 5, purified fusion protein derived from plasmid MBP/VP4LZP expressing the mutant (L537P) leucine zipper region.

    Techniques Used: SDS Page, Mutagenesis, Purification, Derivative Assay, Plasmid Preparation, Expressing

    3) Product Images from "Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly"

    Article Title: Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2013.08.002

    Dppa2 inhibits microtubule assembly around chromatin and in vitro (A) Dppa2 inhibits sperm aster microtubule assembly. Demembranated sperm nuclei were added together with calcium to metaphase extracts supplemented with rhodamine-labeled tubulin (red). Samples were fixed and stained with Hoechst 33342 (blue). (B) Quantification of tubulin fluorescence intensity from (A). Data shown indicate mean and standard error from > 30 asters per sample and are representative of 3 independent experiments. (C) Dppa2 inhibits spindle assembly in a dose-dependent manner. Metaphase spindles were assembled in extracts supplemented with MBP-Dppa2 fusion proteins and rhodamine-labeled tubulin. (D) Quantification of spindle length from (C). Data shown are mean and standard deviation from 30 spindles per sample. (E) Chromatin-independent microtubule assembly in Xenopus egg extracts. Recombinant GST-Dppa2 protein was added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (F) Dppa2 inhibits microtubule polymerization in vitro . MBP-Dppa2 was added to purified bovine tubulin together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. Scale bars, 10 μm. .
    Figure Legend Snippet: Dppa2 inhibits microtubule assembly around chromatin and in vitro (A) Dppa2 inhibits sperm aster microtubule assembly. Demembranated sperm nuclei were added together with calcium to metaphase extracts supplemented with rhodamine-labeled tubulin (red). Samples were fixed and stained with Hoechst 33342 (blue). (B) Quantification of tubulin fluorescence intensity from (A). Data shown indicate mean and standard error from > 30 asters per sample and are representative of 3 independent experiments. (C) Dppa2 inhibits spindle assembly in a dose-dependent manner. Metaphase spindles were assembled in extracts supplemented with MBP-Dppa2 fusion proteins and rhodamine-labeled tubulin. (D) Quantification of spindle length from (C). Data shown are mean and standard deviation from 30 spindles per sample. (E) Chromatin-independent microtubule assembly in Xenopus egg extracts. Recombinant GST-Dppa2 protein was added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (F) Dppa2 inhibits microtubule polymerization in vitro . MBP-Dppa2 was added to purified bovine tubulin together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. Scale bars, 10 μm. .

    Techniques Used: In Vitro, Labeling, Staining, Fluorescence, Standard Deviation, Recombinant, Purification

    Dppa2 requires its C terminus but not DNA-binding to inhibit microtubule assembly (A) Schematic of Dppa2 deletion constructs used. (B) Inhibition of spindle assembly requires the Dppa2 C terminus but not DNA-binding. Metaphase spindles were assembled in extracts supplemented with 2 μM MBP-Dppa2 fusion proteins. MBP-Dppa2 localization was visualized by immunofluorescence using an anti-MBP antibody. Scale bar, 10 μm. (C) Quantification of spindle length in (B). Bars represent mean and standard error from 30 spindles and are representative of 3 independent experiments. (D) Chromatin-independent microtubule assembly in Xenopus egg extracts. MBP-Dppa2 proteins were added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (E) Polymerization of purified tubulin in vitro . Purified bovine tubulin was treated with MBP-Dppa2 proteins and 0.5 % DMSO, and polymerized microtubules analyzed by Coomassie staining.
    Figure Legend Snippet: Dppa2 requires its C terminus but not DNA-binding to inhibit microtubule assembly (A) Schematic of Dppa2 deletion constructs used. (B) Inhibition of spindle assembly requires the Dppa2 C terminus but not DNA-binding. Metaphase spindles were assembled in extracts supplemented with 2 μM MBP-Dppa2 fusion proteins. MBP-Dppa2 localization was visualized by immunofluorescence using an anti-MBP antibody. Scale bar, 10 μm. (C) Quantification of spindle length in (B). Bars represent mean and standard error from 30 spindles and are representative of 3 independent experiments. (D) Chromatin-independent microtubule assembly in Xenopus egg extracts. MBP-Dppa2 proteins were added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (E) Polymerization of purified tubulin in vitro . Purified bovine tubulin was treated with MBP-Dppa2 proteins and 0.5 % DMSO, and polymerized microtubules analyzed by Coomassie staining.

    Techniques Used: Binding Assay, Construct, Inhibition, Immunofluorescence, Staining, Purification, In Vitro

    4) Product Images from "Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains"

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Journal: bioRxiv

    doi: 10.1101/113142

    Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.
    Figure Legend Snippet: Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Techniques Used: Western Blot, Construct

    Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.
    Figure Legend Snippet: Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Techniques Used: Western Blot, Expressing, Construct

    5) Product Images from "Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase"

    Article Title: Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

    Journal: eLife

    doi: 10.7554/eLife.59222

    Nud1 and Nud1 7A characterization in vivo and in vitro. ( A ) Nud1 WT -HA 3 and Nud1 7A -HA 3 levels assessed by western blot analysis of cell extracts prepared from asynchronous cultures of the parent strains analyzed in Figure 5 . ( B ) Kinase assay using 4 µg of purified MBP-Nud1 WT or MBP-Nud1 7A and 19 ng of purified Twin-Strep-Clb5 ∆db /Cdc28-as/Cks1 was carried out as described under Materials and methods. 5 µM 1NM-PP1 was added to inhibit Cdc28-as (analog-sensitive CDK). Reactions were resolved by SDS-PAGE and blotted onto a PVDF membrane (bottom) that was exposed to film overnight (top) ( C ) CDK sites in Nud1 contribute to bulk phosphorylation in vivo. Western blot analysis of whole cell extracts from cells expressing Nud1 WT -HA 3 or Nud1 7A -HA 3 before or after depletion of Cdc5 under the control of the repressible MET3 promoter was carried out as described under Materials and methods. Cdc5 also contributes to Nud1 mobility shift by phosphorylation. A further decrease in mobility shift was observed in cells expressing Nud1 7A versus wild type Nud1 following Cdc5 depletion. Linescans along the direction of electrophoretic migration show the relative down-shift of Nud1 7A -HA 3 (in red). ( D ) A Nud1 domain between amino acid positions 250–600 was necessary and sufficient for Spc72 binding in vitro. Previous yeast two-hybrid studies suggested that the Nud1 C-terminus (residues 405–852) associated with Spc72 ( Gruneberg, 2000 ). ( E ) Spc72 pull down by Nud1(250-600) showing enhanced binding following CDK phosphorylation as described for full-length Nud1 in Figure 5J .
    Figure Legend Snippet: Nud1 and Nud1 7A characterization in vivo and in vitro. ( A ) Nud1 WT -HA 3 and Nud1 7A -HA 3 levels assessed by western blot analysis of cell extracts prepared from asynchronous cultures of the parent strains analyzed in Figure 5 . ( B ) Kinase assay using 4 µg of purified MBP-Nud1 WT or MBP-Nud1 7A and 19 ng of purified Twin-Strep-Clb5 ∆db /Cdc28-as/Cks1 was carried out as described under Materials and methods. 5 µM 1NM-PP1 was added to inhibit Cdc28-as (analog-sensitive CDK). Reactions were resolved by SDS-PAGE and blotted onto a PVDF membrane (bottom) that was exposed to film overnight (top) ( C ) CDK sites in Nud1 contribute to bulk phosphorylation in vivo. Western blot analysis of whole cell extracts from cells expressing Nud1 WT -HA 3 or Nud1 7A -HA 3 before or after depletion of Cdc5 under the control of the repressible MET3 promoter was carried out as described under Materials and methods. Cdc5 also contributes to Nud1 mobility shift by phosphorylation. A further decrease in mobility shift was observed in cells expressing Nud1 7A versus wild type Nud1 following Cdc5 depletion. Linescans along the direction of electrophoretic migration show the relative down-shift of Nud1 7A -HA 3 (in red). ( D ) A Nud1 domain between amino acid positions 250–600 was necessary and sufficient for Spc72 binding in vitro. Previous yeast two-hybrid studies suggested that the Nud1 C-terminus (residues 405–852) associated with Spc72 ( Gruneberg, 2000 ). ( E ) Spc72 pull down by Nud1(250-600) showing enhanced binding following CDK phosphorylation as described for full-length Nud1 in Figure 5J .

    Techniques Used: In Vivo, In Vitro, Western Blot, Kinase Assay, Purification, SDS Page, Expressing, Mobility Shift, Migration, Binding Assay

    6) Product Images from "A spatiotemporal molecular switch governs plant asymmetric cell division"

    Article Title: A spatiotemporal molecular switch governs plant asymmetric cell division

    Journal: Nature plants

    doi: 10.1038/s41477-021-00906-0

    BSL1 Physically Interacts with YDA in vitro and in vivo . a , In vitro pull-down assays using purified recombinant proteins show GST-BSL1 interaction with MBP-YDA. MBP-YDA used as bait. GST (negative control) or GST-BSL1 interaction were detected by anti-GST. b , Co-IP data show in vivo interaction between BSL1 and YDA. BSL1-FLAG was used as bait to detect the binding of YDA KI -Myc in 5-dpg Arabidopsis seedlings. The expression of BSL1-FLAG is driven by the TMM promoter and the expression of YDA is driven by the BASL promoter. YDA KI (Kinase Inactive YDA) was used to avoid the activity of YDA strongly suppressing plant growth. Data represent results of experiments repeated three times in a and b . c , BiFC assays show the interaction between BSL1 and YDA KI (kinase inactive YDA variant used to suppress active YDA-triggered cell death) occurs at the cell membrane in N. benthamiana leaf epidermis. Positive protein-protein interactions were visualized by YFP signal (yellow). Half YFPs (nYFP or cYFP) were uses as negative controls. Scale bars, 50 μm.
    Figure Legend Snippet: BSL1 Physically Interacts with YDA in vitro and in vivo . a , In vitro pull-down assays using purified recombinant proteins show GST-BSL1 interaction with MBP-YDA. MBP-YDA used as bait. GST (negative control) or GST-BSL1 interaction were detected by anti-GST. b , Co-IP data show in vivo interaction between BSL1 and YDA. BSL1-FLAG was used as bait to detect the binding of YDA KI -Myc in 5-dpg Arabidopsis seedlings. The expression of BSL1-FLAG is driven by the TMM promoter and the expression of YDA is driven by the BASL promoter. YDA KI (Kinase Inactive YDA) was used to avoid the activity of YDA strongly suppressing plant growth. Data represent results of experiments repeated three times in a and b . c , BiFC assays show the interaction between BSL1 and YDA KI (kinase inactive YDA variant used to suppress active YDA-triggered cell death) occurs at the cell membrane in N. benthamiana leaf epidermis. Positive protein-protein interactions were visualized by YFP signal (yellow). Half YFPs (nYFP or cYFP) were uses as negative controls. Scale bars, 50 μm.

    Techniques Used: In Vitro, In Vivo, Purification, Recombinant, Negative Control, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Activity Assay, Bimolecular Fluorescence Complementation Assay, Variant Assay

    BSL1 Interferes the Interaction of BIN2-BASL in vitro . a , In vitro pull-down assays using recombinant proteins to test the BIN2-BASL interaction in the presence of an increasing amount of MBP-BSL1. His-BASL was used as bait and the amount of GST-BIN2 being pulled down reflects the interaction strength of BIN2-BASL. MBP was used as negative control. Histograms (below) show quantification of relative protein levels of BIN2 in the assay above. Results suggest the addition of MBP-BSL1 reduced the amount of BIN2 that interacted with BASL. Data are presented as mean ± SD. n = three independent experiments. Two-tailed Student’s t-tests. ** P
    Figure Legend Snippet: BSL1 Interferes the Interaction of BIN2-BASL in vitro . a , In vitro pull-down assays using recombinant proteins to test the BIN2-BASL interaction in the presence of an increasing amount of MBP-BSL1. His-BASL was used as bait and the amount of GST-BIN2 being pulled down reflects the interaction strength of BIN2-BASL. MBP was used as negative control. Histograms (below) show quantification of relative protein levels of BIN2 in the assay above. Results suggest the addition of MBP-BSL1 reduced the amount of BIN2 that interacted with BASL. Data are presented as mean ± SD. n = three independent experiments. Two-tailed Student’s t-tests. ** P

    Techniques Used: In Vitro, Recombinant, Negative Control, Two Tailed Test

    The association of BSL1 with the polarity complex activates YDA and MAPK signalling. a , In vitro pull-down experiments showing that BSL1 interacts with YDA. BSL1–FLAG and YDAKI–YFP (kinase was made inactive to avoid overexpression-triggered cell death) were overexpressed in N. benthamiana leaves and purified from cell extracts. The BSL1–YDA interaction was assayed by the amount of BSL1 pulled down by YDA KI –YFP bound to GFP-Trap agarose beads. BSU1–FLAG and YFP alone were used as controls. b , In vitro kinase assays showing that BSL1 directly dephosphorylates YDA. Recombinant proteins of MBP–YDA were produced and purified from E. coli cells. BSL1/BSL1 D584N –FLAG were produced and purified from N. benthamiana leaf cells. Autophosphorylation of YDA was assayed in the presence and absence of BSL1/BSL1 D584N –FLAG, and the phosphorylation levels of YDA were analysed by Phos-tag PAGE. Slow migration indicates that the protein contains more phosphoryl groups. The protein amount used for the assay was visualized by immunoblotting. c , Western blots showing elevated levels of MAPK signalling in vivo. Top: schematic showing that BSL1-mediated dephosphorylation of YDA may activate YDA and promote MAPK signalling in vivo. Bottom: blots showing the phosphorylated MPK3 and MPK6 (activity) levels in 3-d.p.g. Arabidopsis seedlings detected by anti-phospho-p42/p44 (top) and protein loading is shown by Ponceau S staining (bottom). For a – c , results represent three biological replicates.
    Figure Legend Snippet: The association of BSL1 with the polarity complex activates YDA and MAPK signalling. a , In vitro pull-down experiments showing that BSL1 interacts with YDA. BSL1–FLAG and YDAKI–YFP (kinase was made inactive to avoid overexpression-triggered cell death) were overexpressed in N. benthamiana leaves and purified from cell extracts. The BSL1–YDA interaction was assayed by the amount of BSL1 pulled down by YDA KI –YFP bound to GFP-Trap agarose beads. BSU1–FLAG and YFP alone were used as controls. b , In vitro kinase assays showing that BSL1 directly dephosphorylates YDA. Recombinant proteins of MBP–YDA were produced and purified from E. coli cells. BSL1/BSL1 D584N –FLAG were produced and purified from N. benthamiana leaf cells. Autophosphorylation of YDA was assayed in the presence and absence of BSL1/BSL1 D584N –FLAG, and the phosphorylation levels of YDA were analysed by Phos-tag PAGE. Slow migration indicates that the protein contains more phosphoryl groups. The protein amount used for the assay was visualized by immunoblotting. c , Western blots showing elevated levels of MAPK signalling in vivo. Top: schematic showing that BSL1-mediated dephosphorylation of YDA may activate YDA and promote MAPK signalling in vivo. Bottom: blots showing the phosphorylated MPK3 and MPK6 (activity) levels in 3-d.p.g. Arabidopsis seedlings detected by anti-phospho-p42/p44 (top) and protein loading is shown by Ponceau S staining (bottom). For a – c , results represent three biological replicates.

    Techniques Used: In Vitro, Over Expression, Purification, Recombinant, Produced, Polyacrylamide Gel Electrophoresis, Migration, Western Blot, In Vivo, De-Phosphorylation Assay, Activity Assay, Staining

    BSLf Physically Interact with BASL in Plants. a-b , Co-IP assays using purified fusion proteins that are transiently overexpressed (driven by the ubiquitous 35S promoter) in N. benthamiana leaf cells. a , Results show physical association of FLAG-tagged BSL1/2/3 proteins with GFP-BASL. GFP-BASL (right) or GFP (left, control) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats. b , Results show physical association of FLAG-tagged BSL1 D584N (catalytically inactive BSL1 variant) proteins with GFP-BASL and BIN2-YFP. GFP alone (left, control), GFP-BASL (middle) or BIN2-YFP (right) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats.
    Figure Legend Snippet: BSLf Physically Interact with BASL in Plants. a-b , Co-IP assays using purified fusion proteins that are transiently overexpressed (driven by the ubiquitous 35S promoter) in N. benthamiana leaf cells. a , Results show physical association of FLAG-tagged BSL1/2/3 proteins with GFP-BASL. GFP-BASL (right) or GFP (left, control) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats. b , Results show physical association of FLAG-tagged BSL1 D584N (catalytically inactive BSL1 variant) proteins with GFP-BASL and BIN2-YFP. GFP alone (left, control), GFP-BASL (middle) or BIN2-YFP (right) was used as bait to bind to the GFP-Trap agarose. Immunoprecipitated proteins were detected by anti-FLAG. Data represent results of three biological repeats.

    Techniques Used: Co-Immunoprecipitation Assay, Purification, Immunoprecipitation, Variant Assay

    7) Product Images from "The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases"

    Article Title: The unusual structure of the PiggyMac cysteine-rich domain reveals zinc finger diversity in PiggyBac-related transposases

    Journal: Mobile DNA

    doi: 10.1186/s13100-021-00240-4

    Interaction of the Pgm CRD with histone H3. a In vitro pulldown assay of P. tetraurelia histones with GST or GST-Pgm(692–768) (GST-CRD). b In vitro pulldown assay of P. tetraurelia histones with MBP or MBP-Pgm(692–768) (MBP-CRD). MBP fusion proteins and histone H3 were revealed on western blots using α-MBP-HRP and α-H3 antibodies, respectively (see Fig. S4 for the control of histone preparations and full-size blots with molecular weight markers). c Amino acid composition of the synthetic peptides used in this study. Methylated lysines are underlined. Hydrophobic residues are in red, residues with positively charged side chains in blue, residues with uncharged polar side chains in purple. C-terminally biotinylated peptides (−b) used in ELISA assays are indicated. d ELISA assays with 100 nM MBP or MBP-Pgm(692–768) (MBP-CRD) against unmodified H3(1–19), trimethylated H3(1–19)K4me3 and H3(1–19)K9m3, control peptides Scrambled 1 (*: TECAN absorbance detection at 450 nm close to saturation) and Scrambled 2, unmodified H3(16–35) and trimethylated H3(16–35)K27me3. p -values were calculated using the Wilcoxon-Mann-Whitney test calculator ( https://ccb-compute2.cs.uni-saarland.de/wtest/ ); sample sizes m = 3, n = 3 and test variant H1: (H3(1–19)K4me3 signal)
    Figure Legend Snippet: Interaction of the Pgm CRD with histone H3. a In vitro pulldown assay of P. tetraurelia histones with GST or GST-Pgm(692–768) (GST-CRD). b In vitro pulldown assay of P. tetraurelia histones with MBP or MBP-Pgm(692–768) (MBP-CRD). MBP fusion proteins and histone H3 were revealed on western blots using α-MBP-HRP and α-H3 antibodies, respectively (see Fig. S4 for the control of histone preparations and full-size blots with molecular weight markers). c Amino acid composition of the synthetic peptides used in this study. Methylated lysines are underlined. Hydrophobic residues are in red, residues with positively charged side chains in blue, residues with uncharged polar side chains in purple. C-terminally biotinylated peptides (−b) used in ELISA assays are indicated. d ELISA assays with 100 nM MBP or MBP-Pgm(692–768) (MBP-CRD) against unmodified H3(1–19), trimethylated H3(1–19)K4me3 and H3(1–19)K9m3, control peptides Scrambled 1 (*: TECAN absorbance detection at 450 nm close to saturation) and Scrambled 2, unmodified H3(16–35) and trimethylated H3(16–35)K27me3. p -values were calculated using the Wilcoxon-Mann-Whitney test calculator ( https://ccb-compute2.cs.uni-saarland.de/wtest/ ); sample sizes m = 3, n = 3 and test variant H1: (H3(1–19)K4me3 signal)

    Techniques Used: In Vitro, Western Blot, Molecular Weight, Methylation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Variant Assay

    8) Product Images from "The Salmonella Typhimurium InvF-SicA complex is necessary for the transcription of sopB in the absence of the repressor H-NS"

    Article Title: The Salmonella Typhimurium InvF-SicA complex is necessary for the transcription of sopB in the absence of the repressor H-NS

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0240617

    InvF does not form dimers. Plasmids expressing either MBP or MBP-InvF were transformed into a Salmonella invF ::Flag strain and samples were collected from IPTG and SPI-1 induced cultures. Cell free extracts were prepared, pulled down with amylose resin to detect interactions of MBP and MBP-InvF and tested by Western blot. In (A) pull downs were tested to detect production of both MBP versions with an anti-MBP antibody (Input). In (B) an anti-Flag antibody was used to detect possible interactions of InvF-Flag with MBP and MBP-InvF (Affinity purified). As a control for the expression of InvF-Flag in the tested conditions the last lane shows a whole cell extract (Input).
    Figure Legend Snippet: InvF does not form dimers. Plasmids expressing either MBP or MBP-InvF were transformed into a Salmonella invF ::Flag strain and samples were collected from IPTG and SPI-1 induced cultures. Cell free extracts were prepared, pulled down with amylose resin to detect interactions of MBP and MBP-InvF and tested by Western blot. In (A) pull downs were tested to detect production of both MBP versions with an anti-MBP antibody (Input). In (B) an anti-Flag antibody was used to detect possible interactions of InvF-Flag with MBP and MBP-InvF (Affinity purified). As a control for the expression of InvF-Flag in the tested conditions the last lane shows a whole cell extract (Input).

    Techniques Used: Expressing, Transformation Assay, Western Blot, Affinity Purification

    9) Product Images from "LEUNIG_HOMOLOG Mediates MYC2-Dependent Transcriptional Activation in Cooperation with the Coactivators HAC1 and MED25 [OPEN]"

    Article Title: LEUNIG_HOMOLOG Mediates MYC2-Dependent Transcriptional Activation in Cooperation with the Coactivators HAC1 and MED25 [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.19.00115

    MED25 Interacts with LUH and LUG, but Not with TPL. (A) Y2H assays examining interactions between the GAL4 DNA AD fusions of LUH, LUG, TPL, and MYC2, and GAL4 DNA BD fusions of MED25, LUH, and LUG. The transformed yeast cells were plated on SD medium lacking His, Ade, Leu, and Trp (SD/-4). The empty AD vector was used as a negative control. (B) In vitro pull-down assays to verify the interaction between LUH and MED25. Purified MBP-LUH protein was incubated with GST or GST-MED25 MA (MD and ACID domains) for the GST pull-down assay and detected by immunoblotting using an anti-MBP antibody (top). The positions of puri?ed GST and GST-MED25 MA proteins separated by SDS-PAGE are marked with asterisks on the Coomassie blue–stained gel (bottom). (C) and (D) Co-IP assays to verify in vivo interactions between LUH and MED25 using 10-d-old MED25-myc seedlings (C) and between LUH and MYC2 using 10-d-old MYC2-myc seedlings (D) . Seedlings were treated with 0.1% (v/v) ethanol for 60 min (mock, M) or 100 μM MeJA for the indicated times. The wild-type (WT) seedlings were used as negative controls. Protein from each sample was immunoprecipitated using an anti-myc antibody and immunoblotted using an anti-LUH antibody. Bands were quantified using ImageJ software, and levels relative to the mock control are shown under each band. All experiments in (A) to (D) were repeated at least three times with similar results. IP, immunoprecipitation.
    Figure Legend Snippet: MED25 Interacts with LUH and LUG, but Not with TPL. (A) Y2H assays examining interactions between the GAL4 DNA AD fusions of LUH, LUG, TPL, and MYC2, and GAL4 DNA BD fusions of MED25, LUH, and LUG. The transformed yeast cells were plated on SD medium lacking His, Ade, Leu, and Trp (SD/-4). The empty AD vector was used as a negative control. (B) In vitro pull-down assays to verify the interaction between LUH and MED25. Purified MBP-LUH protein was incubated with GST or GST-MED25 MA (MD and ACID domains) for the GST pull-down assay and detected by immunoblotting using an anti-MBP antibody (top). The positions of puri?ed GST and GST-MED25 MA proteins separated by SDS-PAGE are marked with asterisks on the Coomassie blue–stained gel (bottom). (C) and (D) Co-IP assays to verify in vivo interactions between LUH and MED25 using 10-d-old MED25-myc seedlings (C) and between LUH and MYC2 using 10-d-old MYC2-myc seedlings (D) . Seedlings were treated with 0.1% (v/v) ethanol for 60 min (mock, M) or 100 μM MeJA for the indicated times. The wild-type (WT) seedlings were used as negative controls. Protein from each sample was immunoprecipitated using an anti-myc antibody and immunoblotted using an anti-LUH antibody. Bands were quantified using ImageJ software, and levels relative to the mock control are shown under each band. All experiments in (A) to (D) were repeated at least three times with similar results. IP, immunoprecipitation.

    Techniques Used: Transformation Assay, Plasmid Preparation, Negative Control, In Vitro, Purification, Incubation, Pull Down Assay, SDS Page, Staining, Co-Immunoprecipitation Assay, In Vivo, Immunoprecipitation, Software

    10) Product Images from "A feminizing switch in a hemimetabolous insect"

    Article Title: A feminizing switch in a hemimetabolous insect

    Journal: Science Advances

    doi: 10.1126/sciadv.abf9237

    Effect of Nlfmd-F and Nlfmd2 340 products on the splicing patterns of Nldsx and Nldsx-202 pre-mRNA in 293T cells. ( A ) Mini-genes and its default splicing in 293T cells. Boxes and the lines between boxes represent the exon and intron sequences, respectively. In the p3XFlag- Nldsx-202 , a region of 202 bp was deleted (indicated by black box), and the sequence was listed below in gray box from the female-specific exon 6f. ( B ) Copy number of pm Nldsx-F and pm Nldsx-M by absolute quantification in real-time qPCR. ( C ) The splicing of p3XFlag- Nldsx and p3XFlag- Nldsx-202 was regulated differently by Nlfmd-F and Nlfmd2 340 . (c1) The specific primers of pm Nldsx-F and pm Nldsx-M were used to test the splicing changes by semiquantitative RT-PCR. (c2) The grayscale value of the pm Nldsx-M , pm Nldsx-M+F-1 , and pm Nldsx-M+F-2 bands was measured by software Image J. (c3) The expressions of Nldsx-F were measured by absolute quantification with primer pm Nldsx-F . (c4) Different model of Nldsx alternative splicing in 293T cells. Naming of samples 1 to 8 is consistent in (c1), (c2), and (c3). ( D ) NlFMD-F immunoprecipitated (IP) with Nlfmd 2 340 . ( E ) NlFMD-Flag pull-down assay with NlFMD2 340 -Mbp. Data in (B) are represented as means ± SEM. Student’s t test was used in (B) (** P
    Figure Legend Snippet: Effect of Nlfmd-F and Nlfmd2 340 products on the splicing patterns of Nldsx and Nldsx-202 pre-mRNA in 293T cells. ( A ) Mini-genes and its default splicing in 293T cells. Boxes and the lines between boxes represent the exon and intron sequences, respectively. In the p3XFlag- Nldsx-202 , a region of 202 bp was deleted (indicated by black box), and the sequence was listed below in gray box from the female-specific exon 6f. ( B ) Copy number of pm Nldsx-F and pm Nldsx-M by absolute quantification in real-time qPCR. ( C ) The splicing of p3XFlag- Nldsx and p3XFlag- Nldsx-202 was regulated differently by Nlfmd-F and Nlfmd2 340 . (c1) The specific primers of pm Nldsx-F and pm Nldsx-M were used to test the splicing changes by semiquantitative RT-PCR. (c2) The grayscale value of the pm Nldsx-M , pm Nldsx-M+F-1 , and pm Nldsx-M+F-2 bands was measured by software Image J. (c3) The expressions of Nldsx-F were measured by absolute quantification with primer pm Nldsx-F . (c4) Different model of Nldsx alternative splicing in 293T cells. Naming of samples 1 to 8 is consistent in (c1), (c2), and (c3). ( D ) NlFMD-F immunoprecipitated (IP) with Nlfmd 2 340 . ( E ) NlFMD-Flag pull-down assay with NlFMD2 340 -Mbp. Data in (B) are represented as means ± SEM. Student’s t test was used in (B) (** P

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Software, Immunoprecipitation, Pull Down Assay

    11) Product Images from "The role of OsMSH4 in male and female gamete development in rice meiosis"

    Article Title: The role of OsMSH4 in male and female gamete development in rice meiosis

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erv540

    OsMSH5 interacts with OsMSH4, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c. (A) Yeast-two-hybrid (Y2H) assays to test the interaction region between OsMSH4 and OsMSH5. Constructs expressing different regions of OsMSH4 and mutated Osmsh4 were prepared in the bait vector pGBKT7 (BD) (left). Numbers indicate amino acid residues, and the red line indicates mutated amino acids. Full-length OsMSH5 was cloned into the prey vector pGADT7 (AD). -LTH, selective medium (SD–Leu/–Trp/–His); -LTHA, selective medium (SD–Leu/–Trp/–His/–Ade). (B) In vitro pull-down assay of recombinant glutathione S -transferase (GST)–OsMSH5 using bead-coupled maltose-binding protein (MBP)–OsMSH4 and MBP–Osmsh4. (C) Y2H assay to test interactions between OsMSH5, OsRPA1c, and OsRPA2c. OsMSH5 was cloned into the prey vector pGADT7 (AD), and OsRPA1c and OsRPA2c were inserted in the bait vector pGBKT7 (BD). (D) In vitro pull-down assay of recombinant MBP–OsRPA1c and MBP–OsRPA2c using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA1c and OsMBP–OsRPA2c proteins, respectively. (E) Y2H assay to test interactions between OsMSH5, OsRPA1a, and OsRPA2b. (F) In vitro pull-down assay of recombinant MBP–OsRPA1a using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA1a. (G) In vitro pull-down assay of recombinant MBP–OsRPA2b using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA2b.
    Figure Legend Snippet: OsMSH5 interacts with OsMSH4, OsRPA1a, OsRPA2b, OsRPA1c, and OsRPA2c. (A) Yeast-two-hybrid (Y2H) assays to test the interaction region between OsMSH4 and OsMSH5. Constructs expressing different regions of OsMSH4 and mutated Osmsh4 were prepared in the bait vector pGBKT7 (BD) (left). Numbers indicate amino acid residues, and the red line indicates mutated amino acids. Full-length OsMSH5 was cloned into the prey vector pGADT7 (AD). -LTH, selective medium (SD–Leu/–Trp/–His); -LTHA, selective medium (SD–Leu/–Trp/–His/–Ade). (B) In vitro pull-down assay of recombinant glutathione S -transferase (GST)–OsMSH5 using bead-coupled maltose-binding protein (MBP)–OsMSH4 and MBP–Osmsh4. (C) Y2H assay to test interactions between OsMSH5, OsRPA1c, and OsRPA2c. OsMSH5 was cloned into the prey vector pGADT7 (AD), and OsRPA1c and OsRPA2c were inserted in the bait vector pGBKT7 (BD). (D) In vitro pull-down assay of recombinant MBP–OsRPA1c and MBP–OsRPA2c using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA1c and OsMBP–OsRPA2c proteins, respectively. (E) Y2H assay to test interactions between OsMSH5, OsRPA1a, and OsRPA2b. (F) In vitro pull-down assay of recombinant MBP–OsRPA1a using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA1a. (G) In vitro pull-down assay of recombinant MBP–OsRPA2b using bead-coupled GST–OsMSH5. Asterisks indicate the full-length MBP–OsRPA2b.

    Techniques Used: Construct, Expressing, Plasmid Preparation, Clone Assay, In Vitro, Pull Down Assay, Recombinant, Binding Assay, Y2H Assay

    12) Product Images from "HELLS and CDCA7 comprise a bipartite nucleosome remodeling complex defective in ICF syndrome"

    Article Title: HELLS and CDCA7 comprise a bipartite nucleosome remodeling complex defective in ICF syndrome

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

    doi: 10.1073/pnas.1717509115

    CDCA7e directly recruits HELLS to chromatin. ( A and B ) Western blot analysis of CDCA7e ( A ) and HELLS ( B ) immunoprecipitation from M phase Xenopus extracts. Preimmune rabbit IgG was used to control for nonspecific binding. Representative of n = 3 independent experiments. ( C ) Coomassie-stained gel of purified HELLS–CBP coimmunoprecipitation with MBP–CDCA7. Purified HELLS–CBP was incubated with MBP–CDCA7e or MBP alone and immunoisolation was performed using beads coupled with anti-MBP antibodies or control IgG. Representative of n = 2 independent experiments. ( D ) Western blot analyses of proteins copurified with nucleosome beads recovered from interphase extracts mock depleted or depleted of HELLS or CDCA7e. Representative of n = 4 independent experiments. ( E ) Abundance of HELLS and CDCA7e on nucleosome beads recovered from interphase or M phase extracts mock depleted or depleted of HELLS or CDCA7e, quantified by LC-MS/MS. ( F ) Coomassie-stained gel of a pulldown of nucleosome or DNA beads incubated with MBP–CDCA7e. Uncoupled beads were used to control for nonspecific binding. Representative of n = 2 independent experiments.
    Figure Legend Snippet: CDCA7e directly recruits HELLS to chromatin. ( A and B ) Western blot analysis of CDCA7e ( A ) and HELLS ( B ) immunoprecipitation from M phase Xenopus extracts. Preimmune rabbit IgG was used to control for nonspecific binding. Representative of n = 3 independent experiments. ( C ) Coomassie-stained gel of purified HELLS–CBP coimmunoprecipitation with MBP–CDCA7. Purified HELLS–CBP was incubated with MBP–CDCA7e or MBP alone and immunoisolation was performed using beads coupled with anti-MBP antibodies or control IgG. Representative of n = 2 independent experiments. ( D ) Western blot analyses of proteins copurified with nucleosome beads recovered from interphase extracts mock depleted or depleted of HELLS or CDCA7e. Representative of n = 4 independent experiments. ( E ) Abundance of HELLS and CDCA7e on nucleosome beads recovered from interphase or M phase extracts mock depleted or depleted of HELLS or CDCA7e, quantified by LC-MS/MS. ( F ) Coomassie-stained gel of a pulldown of nucleosome or DNA beads incubated with MBP–CDCA7e. Uncoupled beads were used to control for nonspecific binding. Representative of n = 2 independent experiments.

    Techniques Used: Western Blot, Immunoprecipitation, Binding Assay, Staining, Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    13) Product Images from "A mechanism for the suppression of homologous recombination in G1 cells"

    Article Title: A mechanism for the suppression of homologous recombination in G1 cells

    Journal: Nature

    doi: 10.1038/nature16142

    a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a
    Figure Legend Snippet: a, Site-specific chemical ubiquitylation of HA-PALB2 (1-103) at residue 20 (PALB2-K C 20-Ub) and 45 (PALB2-K C 45-Ub) was carried out by dichloroacetone linking. The resulting ubiquitylated PALB2 polypeptides along with their unmodified counterparts were subjected to pulldown with a fusion of MBP with the coiled-coil domain of BRCA1 (MBP-BRCA1-CC). I, input; PD, pulldown. Asterisk (*) indicates a non-specific band. b , Wild-type and KEAP1Δ 293T cells were treated with cycloheximide (CHX) for the indicated time and then processed for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as a loading control. c , Immunoprecipitation (IP) of USP11 from extracts prepared from 293T cells that were or were not treated with camptothecin (CPT; 200 nM). IP with normal IgG was performed as a control. d , U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 h post-transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vectors (WT=wild type; CS= C318S and CA= C318A catalytically-dead mutants) or an empty vector (EV), with or without an I-SceI expression vector. The percentage of GFP-positive cells was determined 48 h post-plasmid transfection for each condition and was normalized to the I-SceI + non-targeting (siCTRL) condition (mean ± s.d., N =3). e , Schematic representation of human USP11 (top) and KEAP1 (bottom) gene organization and targeting sites of sgRNAs (as described in ) used to generate the USP11Δ and USP11Δ / KEAP1Δ 293T cells. The indels introduced by the CRISPR/Cas9 and their respective frequencies are indicated. The USP11 knockout was created first and subsequently used to make the USP11Δ / KEAP1Δ double mutant. f , Immunoprecipitation (IP) of PALB2 from extracts prepared from 293T cells transfected with the indicated siRNA and with or without CPT (200 nM) treatment. IP with normal IgG was performed as a control. Extended Data Figure 1a

    Techniques Used: Immunoprecipitation, Cycling Probe Technology, Transfection, Expressing, Plasmid Preparation, CRISPR, Knock-Out, Mutagenesis

    Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.
    Figure Legend Snippet: Ubiquitylation of PALB2 prevents BRCA1-PALB2 interaction a, Sequence of the PALB2 N-terminus and mutants. b , GFP IP of extracts derived from G1- or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 proteins. c , In vitro ubiquitylation of the indicated HA-tagged PALB2 proteins by CRL3-KEAP1. d , Pulldown assay of ubiquitylated HA-PALB2 (1-103) incubated with MBP or MBP-BRCA1-CC. I: input, PD: pulldown, FT: flow-through. The asterisk denotes a fragment of HA-PALB2 competent for BRCA1 binding.

    Techniques Used: Sequencing, Derivative Assay, Expressing, In Vitro, Incubation, Flow Cytometry, Binding Assay

    14) Product Images from "Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast ▿Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast ▿ †"

    Article Title: Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast ▿Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01529-09

    Interaction of Spn2 and Spn7 with phosphoinositides. (A) Clusters of basic amino acids near the N termini of some septins. Spn1 to Spn4 were aligned with their S. cerevisiae orthologues to show the conservation of this aspect of septin structure. For Spn5 to Spn7 (which do not have clear orthologues), the N-terminal sequences are shown without comparisons. Red, basic amino acids; green, acidic amino acids; numbers, amino acid positions relative to the N termini. (B) Binding of Spn2 and Spn7 to PtdIns(4)P and PtdIns(5)P in a protein-lipid overlay assay. Phosphoinositide spots on a nitrocellulose membrane were incubated with purified MBP or the indicated MBP fusion proteins (see Materials and Methods). The fusion proteins contained N-terminal fragments (136 to 200 amino acids in length) of the respective septin. Bound proteins were detected using an anti-MBP antibody. The positions of lipid spots were as follows: a, PtdIns; b, PtdIns(4)P; c, PtdIns(5)P; d, PtdIns(4,5)P 2 ; e, PtdIns(3)P; f, PtdIns(3,4)P 2 ; g, PtdIns(3,5)P 2 ; and h, PtdIns(3,4,5)P 3 . Small, uneven signals (such as seen for MBP-Spn2N [g]) appear to be nonspecific background because they were independent of the dose of lipid on the membrane (not shown). (C) Presence of PtdIns(4)P in normal FSMs and in the abnormal FSMs of a pik1 mutant. Wild-type strain THP18 (a to c) and pik1 Δ 117-198 strain MO599 (d to f) were transformed with pREP41(GFP-PH Osh2 -dimer), grown in EMM-plus-thiamine liquid medium overnight, shifted to EMM liquid medium (a and d) or to an SSA plate (b, c, e, and f) for 12 h, and examined by fluorescence microscopy. (D) Distinct localizations of septin structures and PtdIns(4)P. Strain MO815 ( spn2 + -mRFP ) was transformed with pREP41(GFP-PH Osh2 -dimer) and treated as in panel C. Maximum projections of deconvoluted images are shown. (E) Formation of septin rings that do not surround the nuclei in sporulating pik1 mutant cells. Strain MO599 ( pik1 Δ 117-198 ) was transformed with plasmid pAL(spn2-GFP), sporulated, and examined by fluorescence microscopy. Cells in metaphase (a) and anaphase (b) and after completion of meiosis II (c) are shown. Arrowhead, a horseshoe-shaped structure that does not surround the adjacent nucleus (seen at 17 to 36% of nuclei at this stage in three independent experiments); arrows, seemingly completed septin rings that do not surround the adjacent nuclei (seen at 76 to 89% of nuclei at this stage in three experiments). (F) Association between abnormal septin rings and abnormal FSMs in pik1 mutant cells. Strain MO832 ( pik1 Δ 117-198 spn2 + -mRFP GFP-psy1 + ) was sporulated and examined by fluorescence microscopy.
    Figure Legend Snippet: Interaction of Spn2 and Spn7 with phosphoinositides. (A) Clusters of basic amino acids near the N termini of some septins. Spn1 to Spn4 were aligned with their S. cerevisiae orthologues to show the conservation of this aspect of septin structure. For Spn5 to Spn7 (which do not have clear orthologues), the N-terminal sequences are shown without comparisons. Red, basic amino acids; green, acidic amino acids; numbers, amino acid positions relative to the N termini. (B) Binding of Spn2 and Spn7 to PtdIns(4)P and PtdIns(5)P in a protein-lipid overlay assay. Phosphoinositide spots on a nitrocellulose membrane were incubated with purified MBP or the indicated MBP fusion proteins (see Materials and Methods). The fusion proteins contained N-terminal fragments (136 to 200 amino acids in length) of the respective septin. Bound proteins were detected using an anti-MBP antibody. The positions of lipid spots were as follows: a, PtdIns; b, PtdIns(4)P; c, PtdIns(5)P; d, PtdIns(4,5)P 2 ; e, PtdIns(3)P; f, PtdIns(3,4)P 2 ; g, PtdIns(3,5)P 2 ; and h, PtdIns(3,4,5)P 3 . Small, uneven signals (such as seen for MBP-Spn2N [g]) appear to be nonspecific background because they were independent of the dose of lipid on the membrane (not shown). (C) Presence of PtdIns(4)P in normal FSMs and in the abnormal FSMs of a pik1 mutant. Wild-type strain THP18 (a to c) and pik1 Δ 117-198 strain MO599 (d to f) were transformed with pREP41(GFP-PH Osh2 -dimer), grown in EMM-plus-thiamine liquid medium overnight, shifted to EMM liquid medium (a and d) or to an SSA plate (b, c, e, and f) for 12 h, and examined by fluorescence microscopy. (D) Distinct localizations of septin structures and PtdIns(4)P. Strain MO815 ( spn2 + -mRFP ) was transformed with pREP41(GFP-PH Osh2 -dimer) and treated as in panel C. Maximum projections of deconvoluted images are shown. (E) Formation of septin rings that do not surround the nuclei in sporulating pik1 mutant cells. Strain MO599 ( pik1 Δ 117-198 ) was transformed with plasmid pAL(spn2-GFP), sporulated, and examined by fluorescence microscopy. Cells in metaphase (a) and anaphase (b) and after completion of meiosis II (c) are shown. Arrowhead, a horseshoe-shaped structure that does not surround the adjacent nucleus (seen at 17 to 36% of nuclei at this stage in three independent experiments); arrows, seemingly completed septin rings that do not surround the adjacent nuclei (seen at 76 to 89% of nuclei at this stage in three experiments). (F) Association between abnormal septin rings and abnormal FSMs in pik1 mutant cells. Strain MO832 ( pik1 Δ 117-198 spn2 + -mRFP GFP-psy1 + ) was sporulated and examined by fluorescence microscopy.

    Techniques Used: Binding Assay, Protein-lipid Overlay Assay (PLOA), Incubation, Purification, Mutagenesis, Transformation Assay, Fluorescence, Microscopy, Plasmid Preparation

    15) Product Images from "The Ska complex promotes Aurora B activity to ensure chromosome biorientation"

    Article Title: The Ska complex promotes Aurora B activity to ensure chromosome biorientation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201603019

    The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).
    Figure Legend Snippet: The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).

    Techniques Used: Activity Assay, In Vitro, Kinase Assay, Staining, Western Blot, Incubation, Recombinant, Autoradiography, Concentration Assay

    16) Product Images from "Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity"

    Article Title: Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity

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

    doi: 10.1073/pnas.2120219119

    Transcription cofactor activity of NBR6. ( A ) Volcano plots illustrating the transcriptome dynamics in double bz1728 , triple bz1728nobiro6 , or single nbr6-t1 mutant in 12-d-old roots. ( B ) Heatmap representation of FC (log 2 FC) for the top 200 most up-regulated or down-regulated genes in bz1728 and bz1728nobiro6 . ( C ) Relative transcript levels for representative UPR-related genes under normal growth conditions, as determined by qRT-PCR. Values in WT were set to 1. ( D ) Schematic diagram of NBR6 and derivatives used in this study. Gray box indicates the eukaryote-conserved HFD. ( E ) Fluorescence signal observed in Arabidopsis protoplasts transiently transfected with constructs encoding sGFP-tagged NBR6 derivatives. ( F ) BiFC signal observed for combinations of constructs encoding C-terminally truncated bZIP60 (bZ60n) and NBR6 derivatives. Constructs were transiently transfected in Arabidopsis protoplasts. DIC, differential interference contrast; H2B-CFP, CFP fluorescence as transfection control and nuclear localization marker; merged, merged image; YFP, YFP fluorescence by BiFC. ( G ) Split-luciferase complementation (split-luc) signal observed from combinations of constructs encoding bZ60n and NBR6 derivatives. Constructs were transiently infiltrated in N. benthamiana leaves. ( H and I ) In vitro pull-down assays between GST-bZ60n and MBP-NBR6 derivatives. Representative immunoblot results of input samples (INPUT) and pulled-down samples with anti-GST antibody (PD-GST) are shown. The asterisk indicates the expected band size for MBP-NBR6n. ( J and K ) Transient transactivation assay for bZ60n and NBR6 derivatives. The schematic diagram of the reporter construct with the BIP3 promoter is shown ( J ). Data are shown with all data points (red circles) and as bar graphs showing the average ± SD from four ( C and K ) or six ( G ) biological replicates. Different letters indicate significant difference (ANOVA post hoc Tukey’s HSD test, P
    Figure Legend Snippet: Transcription cofactor activity of NBR6. ( A ) Volcano plots illustrating the transcriptome dynamics in double bz1728 , triple bz1728nobiro6 , or single nbr6-t1 mutant in 12-d-old roots. ( B ) Heatmap representation of FC (log 2 FC) for the top 200 most up-regulated or down-regulated genes in bz1728 and bz1728nobiro6 . ( C ) Relative transcript levels for representative UPR-related genes under normal growth conditions, as determined by qRT-PCR. Values in WT were set to 1. ( D ) Schematic diagram of NBR6 and derivatives used in this study. Gray box indicates the eukaryote-conserved HFD. ( E ) Fluorescence signal observed in Arabidopsis protoplasts transiently transfected with constructs encoding sGFP-tagged NBR6 derivatives. ( F ) BiFC signal observed for combinations of constructs encoding C-terminally truncated bZIP60 (bZ60n) and NBR6 derivatives. Constructs were transiently transfected in Arabidopsis protoplasts. DIC, differential interference contrast; H2B-CFP, CFP fluorescence as transfection control and nuclear localization marker; merged, merged image; YFP, YFP fluorescence by BiFC. ( G ) Split-luciferase complementation (split-luc) signal observed from combinations of constructs encoding bZ60n and NBR6 derivatives. Constructs were transiently infiltrated in N. benthamiana leaves. ( H and I ) In vitro pull-down assays between GST-bZ60n and MBP-NBR6 derivatives. Representative immunoblot results of input samples (INPUT) and pulled-down samples with anti-GST antibody (PD-GST) are shown. The asterisk indicates the expected band size for MBP-NBR6n. ( J and K ) Transient transactivation assay for bZ60n and NBR6 derivatives. The schematic diagram of the reporter construct with the BIP3 promoter is shown ( J ). Data are shown with all data points (red circles) and as bar graphs showing the average ± SD from four ( C and K ) or six ( G ) biological replicates. Different letters indicate significant difference (ANOVA post hoc Tukey’s HSD test, P

    Techniques Used: Activity Assay, Mutagenesis, Quantitative RT-PCR, Fluorescence, Transfection, Construct, Bimolecular Fluorescence Complementation Assay, Marker, Luciferase, In Vitro, Transactivation Assay

    17) Product Images from "Nicotinamide Adenine Dinucleotide-Dependent Binding of the Neuronal Ca2+ Sensor Protein GCAP2 to Photoreceptor Synaptic Ribbons"

    Article Title: Nicotinamide Adenine Dinucleotide-Dependent Binding of the Neuronal Ca2+ Sensor Protein GCAP2 to Photoreceptor Synaptic Ribbons

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.3701-09.2010

    Coimmunoprecipitation of RIBEYE and GCAP2 from the bovine retina. Aa , GCAP2 immune serum and GCAP2 preimmune serum were tested for their capability to coimmunoprecipitate RIBEYE. The experiments were analyzed by SDS-PAGE (12.5% polyacrylamide gels) followed by Western blotting (WB) with the indicated antibodies. RIBEYE is coimmunoprecipitated by GCAP2 immune serum (lane 2, arrowhead) but not by GCAP2 preimmune serum (lane 1). Ab , The same blot as in Aa but reprobed with rabbit polyclonal anti-GCAP2 antibodies. This blot shows the presence of GCAP2 precipitated by the immune serum (lane 2, arrowhead) but not by the preimmune serum (lane 1). Asterisks indicate the immunoglobulin heavy chains. B , RIBEYE immune serum and RIBEYE preimmune serum were tested for their capability to coimmunoprecipitate GCAP2. The experiments were analyzed by SDS-PAGE (12.5% polyacrylamide gels) followed by Western blotting with the indicated antibodies. GCAP2 is coimmunoprecipitated by RIBEYE immune serum ( Ba , lane 2, arrowhead) but not by RIBEYE preimmune serum ( Ba , lane 1). Bb , The same blot as in Ba but reprobed with polyclonal anti-RIBEYE (U2656). RIBEYE was immunoprecipitated by the RIBEYE immune serum (lane 2, arrowhead) but not by the RIBEYE preimmune serum (lane 1). Asterisks indicate the immunoglobulin heavy chains. Bc , The same blot as in Bb but reprobed with mouse monoclonal anti-CtBP2 antibodies which detect the B domain of RIBEYE. Similar to Bb , this blot also shows the presence of RIBEYE precipitated by the RIBEYE immune serum (lane 2) but not by the RIBEYE preimmune serum (lane 1). In addition to RIBEYE, an additional protein at ~50 kDa is present in the experimental precipitate (lane 2) but not in the control immunoprecipitate (lane 1). This 50 kDa band very likely is CtBP2 ( Bc , lane 2, circle). Purified synaptic ribbons contain RIBEYE and CtBP1 but not CtBP2 (K.S. and F.S., unpublished data). In the input lanes (lane 3), 0.5% of total input was loaded in A , and 1% of total input in B . The immunoprecipitates are always 100%. In the input lanes (“bovine retina lysate”; A , B , lane 3), RIBEYE is only visible as a faint band because RIBEYE is not a major protein in the crude retinal lysate prepared as described in Materials and Methods, and only a limited amount of protein can be loaded on a single lane. The “bovine retina lysate” contains the Triton X-soluble supernatant after tissue extraction and spinning at 13,000 rpm (30 min, 4°C; see Materials and Methods). RIBEYE is strongly enriched in the experimental immunoprecipitates ( Aa , Bb , Bc , lane 2). Lane 4 in Ab serves as positive control. In A , lane 4 is loaded with (total) bovine retina boiled in sample buffer; in B , lane 4 is loaded with purified synaptic ribbons. IP, Immunoprecipitation.
    Figure Legend Snippet: Coimmunoprecipitation of RIBEYE and GCAP2 from the bovine retina. Aa , GCAP2 immune serum and GCAP2 preimmune serum were tested for their capability to coimmunoprecipitate RIBEYE. The experiments were analyzed by SDS-PAGE (12.5% polyacrylamide gels) followed by Western blotting (WB) with the indicated antibodies. RIBEYE is coimmunoprecipitated by GCAP2 immune serum (lane 2, arrowhead) but not by GCAP2 preimmune serum (lane 1). Ab , The same blot as in Aa but reprobed with rabbit polyclonal anti-GCAP2 antibodies. This blot shows the presence of GCAP2 precipitated by the immune serum (lane 2, arrowhead) but not by the preimmune serum (lane 1). Asterisks indicate the immunoglobulin heavy chains. B , RIBEYE immune serum and RIBEYE preimmune serum were tested for their capability to coimmunoprecipitate GCAP2. The experiments were analyzed by SDS-PAGE (12.5% polyacrylamide gels) followed by Western blotting with the indicated antibodies. GCAP2 is coimmunoprecipitated by RIBEYE immune serum ( Ba , lane 2, arrowhead) but not by RIBEYE preimmune serum ( Ba , lane 1). Bb , The same blot as in Ba but reprobed with polyclonal anti-RIBEYE (U2656). RIBEYE was immunoprecipitated by the RIBEYE immune serum (lane 2, arrowhead) but not by the RIBEYE preimmune serum (lane 1). Asterisks indicate the immunoglobulin heavy chains. Bc , The same blot as in Bb but reprobed with mouse monoclonal anti-CtBP2 antibodies which detect the B domain of RIBEYE. Similar to Bb , this blot also shows the presence of RIBEYE precipitated by the RIBEYE immune serum (lane 2) but not by the RIBEYE preimmune serum (lane 1). In addition to RIBEYE, an additional protein at ~50 kDa is present in the experimental precipitate (lane 2) but not in the control immunoprecipitate (lane 1). This 50 kDa band very likely is CtBP2 ( Bc , lane 2, circle). Purified synaptic ribbons contain RIBEYE and CtBP1 but not CtBP2 (K.S. and F.S., unpublished data). In the input lanes (lane 3), 0.5% of total input was loaded in A , and 1% of total input in B . The immunoprecipitates are always 100%. In the input lanes (“bovine retina lysate”; A , B , lane 3), RIBEYE is only visible as a faint band because RIBEYE is not a major protein in the crude retinal lysate prepared as described in Materials and Methods, and only a limited amount of protein can be loaded on a single lane. The “bovine retina lysate” contains the Triton X-soluble supernatant after tissue extraction and spinning at 13,000 rpm (30 min, 4°C; see Materials and Methods). RIBEYE is strongly enriched in the experimental immunoprecipitates ( Aa , Bb , Bc , lane 2). Lane 4 in Ab serves as positive control. In A , lane 4 is loaded with (total) bovine retina boiled in sample buffer; in B , lane 4 is loaded with purified synaptic ribbons. IP, Immunoprecipitation.

    Techniques Used: SDS Page, Western Blot, Immunoprecipitation, Purification, Positive Control

    18) Product Images from "Plant AFC2 kinase desensitizes thermomorphogenesis through modulation of alternative splicing"

    Article Title: Plant AFC2 kinase desensitizes thermomorphogenesis through modulation of alternative splicing

    Journal: iScience

    doi: 10.1016/j.isci.2022.104051

    High temperature reduces AFC2 kinase activity (A) In vitro kinase assay performed at the indicated temperatures. GST-AFC2 kinase and MBP-RSZ21 substrates were incubated at the indicated temperatures with or without ATP for 30 min. Kinase activity was monitored by immunoblotting on Phos-tag SDS-PAGE gels, while the loading controls were detected by immunoblotting on regular SDS-PAGE gels. The shifted MBP-RSZ21 bands on the Phos-tag SDS-PAGE gel represented phosphorylated forms, while the unshifted bands indicated unphosphorylated forms. GST-AFC2 proteins were detected with anti-GST antibody, and MBP-RSZ21 proteins were detected with anti-MBP antibody. (B) Kinase activity reversibility assay. GST-AFC2 kinase and MBP-RSZ21 substrates were initially incubated at the indicated temperatures with or without ATP for 30 min and then stopped by adding sample loading buffer. Two reactions performed at 28°C or 37°C were not stopped but were transferred to 22°C for additional 30 min to test kinase activity reversibility. Kinase activities were then monitored as described in (A).
    Figure Legend Snippet: High temperature reduces AFC2 kinase activity (A) In vitro kinase assay performed at the indicated temperatures. GST-AFC2 kinase and MBP-RSZ21 substrates were incubated at the indicated temperatures with or without ATP for 30 min. Kinase activity was monitored by immunoblotting on Phos-tag SDS-PAGE gels, while the loading controls were detected by immunoblotting on regular SDS-PAGE gels. The shifted MBP-RSZ21 bands on the Phos-tag SDS-PAGE gel represented phosphorylated forms, while the unshifted bands indicated unphosphorylated forms. GST-AFC2 proteins were detected with anti-GST antibody, and MBP-RSZ21 proteins were detected with anti-MBP antibody. (B) Kinase activity reversibility assay. GST-AFC2 kinase and MBP-RSZ21 substrates were initially incubated at the indicated temperatures with or without ATP for 30 min and then stopped by adding sample loading buffer. Two reactions performed at 28°C or 37°C were not stopped but were transferred to 22°C for additional 30 min to test kinase activity reversibility. Kinase activities were then monitored as described in (A).

    Techniques Used: Activity Assay, In Vitro, Kinase Assay, Incubation, SDS Page

    19) Product Images from "The Interaction of Herpes Simplex Virus 1 Regulatory Protein ICP22 with the cdc25C Phosphatase Is Enabled In Vitro by Viral Protein Kinases US3 and UL13 "

    Article Title: The Interaction of Herpes Simplex Virus 1 Regulatory Protein ICP22 with the cdc25C Phosphatase Is Enabled In Vitro by Viral Protein Kinases US3 and UL13

    Journal: Journal of Virology

    doi: 10.1128/JVI.02022-07

    Purified U S 3 and U L 13 protein kinases are active. GST-U S 3 or GST-U S 3-M was reacted with the substrates MBP-cdc25C and MBP-cdc25C-M in the presence of [γ- 32 P]ATP for 30 min. Beads containing the substrate were rinsed, and proteins separated by electrophoresis, transferred, and analyzed by autoradiography (A). The membrane was stained with Ponceau S to detect total protein (C) and then immunoblotted for GST (B). GST-U L 13 and GST-U L 13-M were each reacted with MBP-ICP22, MBP-cdc25C, or MBP-cdc25C-M on beads in the presence of [γ- 32 P]ATP for 30 min. The entire reaction was separated by electrophoresis, transferred to a membrane, and subjected to autoradiography (D). The membrane was stained with Ponceau S to detect total protein (F) and immunoblotted for GST (E). The Ponceau S stain from panel F was darkened (G) to highlight the MBP-ICP22 band, circled.
    Figure Legend Snippet: Purified U S 3 and U L 13 protein kinases are active. GST-U S 3 or GST-U S 3-M was reacted with the substrates MBP-cdc25C and MBP-cdc25C-M in the presence of [γ- 32 P]ATP for 30 min. Beads containing the substrate were rinsed, and proteins separated by electrophoresis, transferred, and analyzed by autoradiography (A). The membrane was stained with Ponceau S to detect total protein (C) and then immunoblotted for GST (B). GST-U L 13 and GST-U L 13-M were each reacted with MBP-ICP22, MBP-cdc25C, or MBP-cdc25C-M on beads in the presence of [γ- 32 P]ATP for 30 min. The entire reaction was separated by electrophoresis, transferred to a membrane, and subjected to autoradiography (D). The membrane was stained with Ponceau S to detect total protein (F) and immunoblotted for GST (E). The Ponceau S stain from panel F was darkened (G) to highlight the MBP-ICP22 band, circled.

    Techniques Used: Purification, Electrophoresis, Autoradiography, Staining

    Purified U S 3 and U L 13 kinases phosphorylate ICP22 and cdc25C. GST-U S 3 or GST-U S 3-M was mixed with GST-U L 13 or GST-U L 13-M in various combinations and added to MBP-ICP22 or MBP-cdc25C-M bound to beads in the presence of [γ- 32 P]ATP for 30 min. Beads were rinsed and proteins separated by electrophoresis, transferred, and analyzed by autoradiography (A). The membrane was stained with Ponceau S to detect total protein (D), immunoblotted first for GST (B), and then reprobed for MBP (C).
    Figure Legend Snippet: Purified U S 3 and U L 13 kinases phosphorylate ICP22 and cdc25C. GST-U S 3 or GST-U S 3-M was mixed with GST-U L 13 or GST-U L 13-M in various combinations and added to MBP-ICP22 or MBP-cdc25C-M bound to beads in the presence of [γ- 32 P]ATP for 30 min. Beads were rinsed and proteins separated by electrophoresis, transferred, and analyzed by autoradiography (A). The membrane was stained with Ponceau S to detect total protein (D), immunoblotted first for GST (B), and then reprobed for MBP (C).

    Techniques Used: Purification, Electrophoresis, Autoradiography, Staining

    20) Product Images from "Accumulation of Heterochromatin Components on the Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus Mediated by the Latency-Associated Nuclear Antigen"

    Article Title: Accumulation of Heterochromatin Components on the Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus Mediated by the Latency-Associated Nuclear Antigen

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.14.7299-7310.2004

    LANA interacts with SUV39H1. (A) Pull-down assay with recombinant MBP-SUV39H1 and its mutants. The constructs are shown on the right. NE from BC3 cells was incubated with 4 μg of MBP-LacZ (lane 2) and with MBP-fused SUV39H1 (lane 3) and its mutants (39-ΔN [aa 113 to 412] [lane 4], 39-SET [aa 250 to 412] [lane 5], and 39-Chromo [aa 1 to 112] [lane 6]). Each precipitate was probed with an anti-LANA antibody (Advanced Biotechnologies Inc.) and an anti-MBP antibody (New England Biolabs Inc.). Asterisks indicate the MBP fusion proteins. WB, Western blotting. (B) Mapping the site of LANA that was required for the interaction with SUV39H1. Truncated LANA mutants (left panel) were expressed in 293 cells and subjected to the pull-down assay described for panel A (1 μg of recombinant proteins was used for each experiment). Each precipitate was probed with an anti-V5 antibody (Invitrogen) to detect wild-type protein (WT), LNΔN, and D1; with a mouse anti-LANA antibody (generated in our laboratory) to detect LANA; and with rabbit polyclonal anti-GFP antibodies (Medical and Biological Laboratory) to detect GFP-N1 and GFP-NE. The asterisk shows a nonspecific band seen in the MBP-LacZ α precipitation.
    Figure Legend Snippet: LANA interacts with SUV39H1. (A) Pull-down assay with recombinant MBP-SUV39H1 and its mutants. The constructs are shown on the right. NE from BC3 cells was incubated with 4 μg of MBP-LacZ (lane 2) and with MBP-fused SUV39H1 (lane 3) and its mutants (39-ΔN [aa 113 to 412] [lane 4], 39-SET [aa 250 to 412] [lane 5], and 39-Chromo [aa 1 to 112] [lane 6]). Each precipitate was probed with an anti-LANA antibody (Advanced Biotechnologies Inc.) and an anti-MBP antibody (New England Biolabs Inc.). Asterisks indicate the MBP fusion proteins. WB, Western blotting. (B) Mapping the site of LANA that was required for the interaction with SUV39H1. Truncated LANA mutants (left panel) were expressed in 293 cells and subjected to the pull-down assay described for panel A (1 μg of recombinant proteins was used for each experiment). Each precipitate was probed with an anti-V5 antibody (Invitrogen) to detect wild-type protein (WT), LNΔN, and D1; with a mouse anti-LANA antibody (generated in our laboratory) to detect LANA; and with rabbit polyclonal anti-GFP antibodies (Medical and Biological Laboratory) to detect GFP-N1 and GFP-NE. The asterisk shows a nonspecific band seen in the MBP-LacZ α precipitation.

    Techniques Used: Pull Down Assay, Recombinant, Construct, Incubation, Western Blot, Generated

    21) Product Images from "Phagocytosis mediated by Yersinia invasin induces collagenase-1 expression in rabbit synovial fibroblasts through a proinflammatory cascade"

    Article Title: Phagocytosis mediated by Yersinia invasin induces collagenase-1 expression in rabbit synovial fibroblasts through a proinflammatory cascade

    Journal: Journal of cell science

    doi:

    Effect of affinity of invasin binding on bead phagocytosis and CL-1 induction. RSF were incubated for 2 or 24 hours with beads coated with MBP (nonspecific binding control), INV497D911E (low-affinity binding) or INV497 (high-affinity binding). Bead phagocytosis was analyzed after 3 hours and CL-1 expression was measured in the supernatant by slot blot after 24 hours. (A) Quantitative representation of bead binding and uptake after 3 hours and CL-1 expression after 24 hours for each type of coating. (B) Micrographs of the phagocytosis assay. The green beads are outside and the red beads are inside the cells. Error bars represent ± s.e.m. of 3 independent experiments. Bar, 50 μm.
    Figure Legend Snippet: Effect of affinity of invasin binding on bead phagocytosis and CL-1 induction. RSF were incubated for 2 or 24 hours with beads coated with MBP (nonspecific binding control), INV497D911E (low-affinity binding) or INV497 (high-affinity binding). Bead phagocytosis was analyzed after 3 hours and CL-1 expression was measured in the supernatant by slot blot after 24 hours. (A) Quantitative representation of bead binding and uptake after 3 hours and CL-1 expression after 24 hours for each type of coating. (B) Micrographs of the phagocytosis assay. The green beads are outside and the red beads are inside the cells. Error bars represent ± s.e.m. of 3 independent experiments. Bar, 50 μm.

    Techniques Used: Binding Assay, Incubation, Expressing, Dot Blot, Phagocytosis Assay

    22) Product Images from "The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins"

    Article Title: The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins

    Journal: The Biochemical journal

    doi: 10.1042/BJ20081449

    Comparison of the electrophoretic mobility of MBP-537 chimaera compared to native TSP
    Figure Legend Snippet: Comparison of the electrophoretic mobility of MBP-537 chimaera compared to native TSP

    Techniques Used:

    Comparison of antibody reactivity of the chimaeric MBP-537 with antibody reactivity of the TSP and monomeric MBP
    Figure Legend Snippet: Comparison of antibody reactivity of the chimaeric MBP-537 with antibody reactivity of the TSP and monomeric MBP

    Techniques Used:

    MBP-537 chimaera refolds following a similar pathway as TSP
    Figure Legend Snippet: MBP-537 chimaera refolds following a similar pathway as TSP

    Techniques Used:

    23) Product Images from "Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains"

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Journal: bioRxiv

    doi: 10.1101/113142

    Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.
    Figure Legend Snippet: Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Techniques Used: Western Blot, Construct

    Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.
    Figure Legend Snippet: Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Techniques Used: Western Blot, Expressing, Construct

    24) Product Images from "A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity"

    Article Title: A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

    Journal: Bioscience Reports

    doi: 10.1042/BSR20160040

    His–MBP–TAT and His–MBP–TAT–AICD successfully penetrate HeLa cells ( A ) A time course of protein uptake into HeLa cells for 5–60 min in which 700 nM His–MBP–TAT, His–MBP–TAT–AICD and His–MBP–AICD were added to the cells and incubated for the times indicated, followed by fixation, permeabilization and immunostaining with an anti-MBP antibody and an anti-mouse Alexa 555 secondary antibody. In His–MBP–TAT and His–MBP–TAT–AICD treated cells strong staining is visible, suggesting that the TAT domain enabled protein uptake into the cells where it was retained. The His–MBP–AICD control protein, lacking the TAT domain failed to show any appreciable staining, demonstrating that the TAT domain is necessary for cell penetration. His–MBP–TAT showed diffuse localization throughout the time course whereas His–MBP–TAT–AICD strongly localized to vesicular structures, particularly so after 30 min and 60 min. The outline of example cells is shown in white to facilitate the interpretation of the images. ( B ) HeLa cells transfected with the PI(3,5) P 2 specific GFP-ML1Nx2 probe were incubated with 700 nM of the fusion proteins indicated for 30 min, followed by fixation, permeabilization and immunostaining with an anti-MBP antibody. His–MBP–TAT–AICD was found to display co-localization with the GFP-ML1Nx2 probe. (A and B) White boxes indicate the area enlarged in the inset. Scale bars, 20 μm.
    Figure Legend Snippet: His–MBP–TAT and His–MBP–TAT–AICD successfully penetrate HeLa cells ( A ) A time course of protein uptake into HeLa cells for 5–60 min in which 700 nM His–MBP–TAT, His–MBP–TAT–AICD and His–MBP–AICD were added to the cells and incubated for the times indicated, followed by fixation, permeabilization and immunostaining with an anti-MBP antibody and an anti-mouse Alexa 555 secondary antibody. In His–MBP–TAT and His–MBP–TAT–AICD treated cells strong staining is visible, suggesting that the TAT domain enabled protein uptake into the cells where it was retained. The His–MBP–AICD control protein, lacking the TAT domain failed to show any appreciable staining, demonstrating that the TAT domain is necessary for cell penetration. His–MBP–TAT showed diffuse localization throughout the time course whereas His–MBP–TAT–AICD strongly localized to vesicular structures, particularly so after 30 min and 60 min. The outline of example cells is shown in white to facilitate the interpretation of the images. ( B ) HeLa cells transfected with the PI(3,5) P 2 specific GFP-ML1Nx2 probe were incubated with 700 nM of the fusion proteins indicated for 30 min, followed by fixation, permeabilization and immunostaining with an anti-MBP antibody. His–MBP–TAT–AICD was found to display co-localization with the GFP-ML1Nx2 probe. (A and B) White boxes indicate the area enlarged in the inset. Scale bars, 20 μm.

    Techniques Used: Incubation, Immunostaining, Staining, Transfection

    25) Product Images from "A conserved regulatory module regulates receptor kinase signaling in immunity and development"

    Article Title: A conserved regulatory module regulates receptor kinase signaling in immunity and development

    Journal: bioRxiv

    doi: 10.1101/2021.01.19.427293

    Conservation of RLK-PBL-POL circuitry in CLEp signaling (A) POL is a substrate of active PBL34. Autoradiogram of in vitro kinase assay incubating equal amounts of MBP-tagged POL with MBP-tagged WT PBL34 or mutant forms of PBL34 (PBL34 D275A or PBL34 L135F ). (B) POL phosphorylation status determines its interaction with CLV1 in planta . CoIP assay of GFP-tagged CLV1 with HA-tagged WT POL or phosphovariants (POL 7A or POL 7D ). ( C ) POL phosphosites control direct interaction with BAM3 in vitro . Amylose pulldown assay using equal amounts of GST-tagged cytosolic domain (CD) of BAM3 with MBP-tagged WT (POL) or phosphomimetic (POL 7D ) variants of POL. (D) PBL34 phosphorylates PLL1 in vitro. In vitro kinase assay incubating equal amounts of MBP-tagged WT version (PBL34) or inactive (PBL34*) of PBL34 recombinant protein with MBP-tagged N-terminus (PLL1-N), catalytically-dead full length (PLL1*-FL), or catalytically-dead C-terminus (PLL1*-C). CBB: Coomassie brilliant blue.
    Figure Legend Snippet: Conservation of RLK-PBL-POL circuitry in CLEp signaling (A) POL is a substrate of active PBL34. Autoradiogram of in vitro kinase assay incubating equal amounts of MBP-tagged POL with MBP-tagged WT PBL34 or mutant forms of PBL34 (PBL34 D275A or PBL34 L135F ). (B) POL phosphorylation status determines its interaction with CLV1 in planta . CoIP assay of GFP-tagged CLV1 with HA-tagged WT POL or phosphovariants (POL 7A or POL 7D ). ( C ) POL phosphosites control direct interaction with BAM3 in vitro . Amylose pulldown assay using equal amounts of GST-tagged cytosolic domain (CD) of BAM3 with MBP-tagged WT (POL) or phosphomimetic (POL 7D ) variants of POL. (D) PBL34 phosphorylates PLL1 in vitro. In vitro kinase assay incubating equal amounts of MBP-tagged WT version (PBL34) or inactive (PBL34*) of PBL34 recombinant protein with MBP-tagged N-terminus (PLL1-N), catalytically-dead full length (PLL1*-FL), or catalytically-dead C-terminus (PLL1*-C). CBB: Coomassie brilliant blue.

    Techniques Used: In Vitro, Kinase Assay, Mutagenesis, Co-Immunoprecipitation Assay, Recombinant

    RLCK-VII-5 isoforms are required for CLE signaling. (A) The pbl34-2 dominant negative allele is less sensitive to exogenous CLEp than the pbl34-3 loss-of-function allele. 7-day-old seedlings grown on media with 50 nM of indicated CLE peptides. NT: not treated. Letters indicate significant differences within the treatments (ANOVA followed by Tukey test). n=11-50. (B) BAM3 and PBL34 transphosphorylate each other in vitro . Autoradiogram of in vitro kinase assay using MBP-tagged WT PBL34 or inactive PBL34 (PBL34*) and GST-tagged WT cytosolic domain (CD) of BAM3 (BAM3-CD) or inactive CD of BAM3 (BAM3*-CD). (C) The L135F mutation disrupts auto- and trans-phosphorylation activity of PBL34. Autoradiogram of in vitro kinase assay incubating equal amounts of GST-tagged BAM3 with MBP-tagged WT PBL34 or mutant forms of PBL34 (PBL34 D275A or PBL34 L135F ). (D) CLV3p responses specifically require the RLCK-VII-5 subfamily. 7-day-old seedlings grown on media with CLV3p as indicated. NT: not treated. Letters indicate significant differences within the treatments (ANOVA followed by Tukey test). n=26-46. (E) RLCK-VII-5 members are expressed in the root with partially overlapping patterns. Confocal microscopy pictures of 6-day-old seedlings carrying PBL34::3xNLS-VENUS, PBL35::3xNLS-VENUS and PBL36::3xNLS - VENUS constructs, respectively in Col-0 background. yellow channel: 3xNLS-VENUS; cyan: propidium iodide cell wall staining. (F-H) PBL34 is expressed in the root, accumulates in the protophloem and localizes to the cytosol and the plasma membrane. Confocal microscopy images of 5-day-old seedlings expressing (G) PBL34-CITRINE fusion protein under control of the PBL34 promoter in the rlck-vii-5 triple mutant. Live imaging; yellow channel: CITRINE; cyan: propidium iodide cell wall staining. (H-I) Immunolocalization of PBL34-GFP protein fusion expressed under control of the PBL34 promoter in pbl34-3 mutant using anti-GFP primary antibody combined with Alexa 546 fluorophore (yellow channel). Cyan: calcofluor white cell wall staining. CBB: Coomassie brilliant blue.
    Figure Legend Snippet: RLCK-VII-5 isoforms are required for CLE signaling. (A) The pbl34-2 dominant negative allele is less sensitive to exogenous CLEp than the pbl34-3 loss-of-function allele. 7-day-old seedlings grown on media with 50 nM of indicated CLE peptides. NT: not treated. Letters indicate significant differences within the treatments (ANOVA followed by Tukey test). n=11-50. (B) BAM3 and PBL34 transphosphorylate each other in vitro . Autoradiogram of in vitro kinase assay using MBP-tagged WT PBL34 or inactive PBL34 (PBL34*) and GST-tagged WT cytosolic domain (CD) of BAM3 (BAM3-CD) or inactive CD of BAM3 (BAM3*-CD). (C) The L135F mutation disrupts auto- and trans-phosphorylation activity of PBL34. Autoradiogram of in vitro kinase assay incubating equal amounts of GST-tagged BAM3 with MBP-tagged WT PBL34 or mutant forms of PBL34 (PBL34 D275A or PBL34 L135F ). (D) CLV3p responses specifically require the RLCK-VII-5 subfamily. 7-day-old seedlings grown on media with CLV3p as indicated. NT: not treated. Letters indicate significant differences within the treatments (ANOVA followed by Tukey test). n=26-46. (E) RLCK-VII-5 members are expressed in the root with partially overlapping patterns. Confocal microscopy pictures of 6-day-old seedlings carrying PBL34::3xNLS-VENUS, PBL35::3xNLS-VENUS and PBL36::3xNLS - VENUS constructs, respectively in Col-0 background. yellow channel: 3xNLS-VENUS; cyan: propidium iodide cell wall staining. (F-H) PBL34 is expressed in the root, accumulates in the protophloem and localizes to the cytosol and the plasma membrane. Confocal microscopy images of 5-day-old seedlings expressing (G) PBL34-CITRINE fusion protein under control of the PBL34 promoter in the rlck-vii-5 triple mutant. Live imaging; yellow channel: CITRINE; cyan: propidium iodide cell wall staining. (H-I) Immunolocalization of PBL34-GFP protein fusion expressed under control of the PBL34 promoter in pbl34-3 mutant using anti-GFP primary antibody combined with Alexa 546 fluorophore (yellow channel). Cyan: calcofluor white cell wall staining. CBB: Coomassie brilliant blue.

    Techniques Used: Dominant Negative Mutation, In Vitro, Kinase Assay, Mutagenesis, Activity Assay, Confocal Microscopy, Construct, Staining, Expressing, Imaging

    26) Product Images from "Interaction between LIS1 and PDE4, and its role in cytoplasmic dynein function"

    Article Title: Interaction between LIS1 and PDE4, and its role in cytoplasmic dynein function

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.082982

    LIS1 interacts directly with PDE4 isoforms. ( A ) Purified MBP or purified recombinant MBP-tagged forms of PDE4B1 and PDE4B2 were incubated with His-tagged purified LIS1 protein. Complexes were immobilised on amylose resin and LIS1 capture detected by immunoblotting.
    Figure Legend Snippet: LIS1 interacts directly with PDE4 isoforms. ( A ) Purified MBP or purified recombinant MBP-tagged forms of PDE4B1 and PDE4B2 were incubated with His-tagged purified LIS1 protein. Complexes were immobilised on amylose resin and LIS1 capture detected by immunoblotting.

    Techniques Used: Purification, Recombinant, Incubation

    Identifying the PDE4D3 interaction sites with LIS1. ( A ) An array of immobilized peptide spots of overlapping 25-mer peptides each shifted along by five amino acids in the entire sequence of the LIS1 was probed for interaction with MBP fusion protein of
    Figure Legend Snippet: Identifying the PDE4D3 interaction sites with LIS1. ( A ) An array of immobilized peptide spots of overlapping 25-mer peptides each shifted along by five amino acids in the entire sequence of the LIS1 was probed for interaction with MBP fusion protein of

    Techniques Used: Sequencing

    27) Product Images from "Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains"

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Journal: bioRxiv

    doi: 10.1101/113142

    Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.
    Figure Legend Snippet: Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Techniques Used: Western Blot, Construct

    Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.
    Figure Legend Snippet: Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Techniques Used: Western Blot, Expressing, Construct

    28) Product Images from "Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor"

    Article Title: Hydrogen peroxide positively regulates brassinosteroid signaling through oxidation of the BRASSINAZOLE-RESISTANT1 transcription factor

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03463-x

    TRXh5 is a redox mediator of BZR1. a BiFC assays showed TRXh5 interaction with BZR1 and BES1 in tobacco leaf cells. Scale bar, 50 μm. b TRXh5 directly interacted with BZR1 in vitro. Glutathione agarose beads containing GST-TRXh5 were incubated with equal amount of MBP or MBP-BZR1. Proteins bound to GST-TRXh5 were detected by immunoblot with anti-MBP antibody. c TRXh5 interacts with BZR1 in plants. Immunoprecipitation was performed using Myc-trap beads and transgenic Arabidopsis plants expressing pBZR1:BZR1-YFP only or co-expressing pBZR1:BZR1-YFP and p35S:TRXh5-Myc , and the immunoblot analyzed using anti-Myc or anti-YFP antibodies. d TRXh5 catalyzed reduction of H 2 O 2 -oxidized BZR1 in vitro. MBP-BZR1 pretreated with H 2 O 2 was incubated with or without a TRX system consisting of TRXh5, NTRA, and NADPH for 3 h at room temperature, and then analyzed by biotin-switch methods. e – g Quantification of QC cell division in the root of trxh5-1 mutant ( e ), TRXh5-Ox ( f ), bzr1-1D and bzr1-1D/TRXh5-Ox ( g ) with or without BL treatment as indicated. At least 50 seedlings were examined for each biological repeat. Error bars represented the s.d. of three independent experiments. * p
    Figure Legend Snippet: TRXh5 is a redox mediator of BZR1. a BiFC assays showed TRXh5 interaction with BZR1 and BES1 in tobacco leaf cells. Scale bar, 50 μm. b TRXh5 directly interacted with BZR1 in vitro. Glutathione agarose beads containing GST-TRXh5 were incubated with equal amount of MBP or MBP-BZR1. Proteins bound to GST-TRXh5 were detected by immunoblot with anti-MBP antibody. c TRXh5 interacts with BZR1 in plants. Immunoprecipitation was performed using Myc-trap beads and transgenic Arabidopsis plants expressing pBZR1:BZR1-YFP only or co-expressing pBZR1:BZR1-YFP and p35S:TRXh5-Myc , and the immunoblot analyzed using anti-Myc or anti-YFP antibodies. d TRXh5 catalyzed reduction of H 2 O 2 -oxidized BZR1 in vitro. MBP-BZR1 pretreated with H 2 O 2 was incubated with or without a TRX system consisting of TRXh5, NTRA, and NADPH for 3 h at room temperature, and then analyzed by biotin-switch methods. e – g Quantification of QC cell division in the root of trxh5-1 mutant ( e ), TRXh5-Ox ( f ), bzr1-1D and bzr1-1D/TRXh5-Ox ( g ) with or without BL treatment as indicated. At least 50 seedlings were examined for each biological repeat. Error bars represented the s.d. of three independent experiments. * p

    Techniques Used: Bimolecular Fluorescence Complementation Assay, In Vitro, Incubation, Immunoprecipitation, Transgenic Assay, Expressing, Mutagenesis

    29) Product Images from "Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda"

    Article Title: Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012910

    Western blot analysis of the localization of EvpC using anti-EvpC antibody. (A) Wild type (B) N-terminal triple mutant (C) C-terminal mutant (D) Periplasmic marker (E) Cytoplasmic marker. EvpC is predominantly localized in the periplasmic space and is secreted outside only by the wild type bacteria. 1. Cytoplasmic fraction 2. Periplasmic fraction 3. Secreted fraction. Anti-MBP Monoclonal antibody (NEB) and Anti-DnaK Monoclonal antibody (Stressgen) were used as periplasmic and cytoplasmic markers.
    Figure Legend Snippet: Western blot analysis of the localization of EvpC using anti-EvpC antibody. (A) Wild type (B) N-terminal triple mutant (C) C-terminal mutant (D) Periplasmic marker (E) Cytoplasmic marker. EvpC is predominantly localized in the periplasmic space and is secreted outside only by the wild type bacteria. 1. Cytoplasmic fraction 2. Periplasmic fraction 3. Secreted fraction. Anti-MBP Monoclonal antibody (NEB) and Anti-DnaK Monoclonal antibody (Stressgen) were used as periplasmic and cytoplasmic markers.

    Techniques Used: Western Blot, Mutagenesis, Marker

    30) Product Images from "BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration"

    Article Title: BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E04-02-0141

    PXXP core motif within the PRR of BPGAP1 is indispensable for binding cortactin. (A) Schematic diagram of BPGAP1 PP mutant, indicating substitution of the proline residues at 184 and 186 with alanines (arrows). (B) GST-recombinants of BPGAP1 FL and point mutant (PP) were used for pulldown assays by using lysates prepared from HeLa cells. Beads were washed and processed for Western analysis by using cortactin antibody. (C) Purified SH3(2) fusion of maltose binding protein, MBP-SH3(2), or the MBP control were incubated with Sepharose beads conjugated with purified GST recombinant proteins of either the full-length wild type or PP mutant of BPGAP1 or GST control, and bound targets revealed by Western blot analyses by using MBP antibody. Purified MBP and MBP-SH3(2) were analyzed with MBP antibody and revealed intact targets used in the direct binding assays. The lower apparent molecular weight for MBP-SH3(2) compared with the MBP alone was due to the removal of internal lacZ coding sequence upon cloning of the target insert. Blot was stripped and stained by amido black to verify loading of equal amounts of GSTs. (D) Cells were transfected with either the wild-type FL or PP mutant of FLAG-tagged BPGAP1 and immunoprecipitated with anti-FLAG M2 beads, washed, and analyzed for bound endogenous cortactin by using cortactin antibody. WCL, whole-cell lysates.
    Figure Legend Snippet: PXXP core motif within the PRR of BPGAP1 is indispensable for binding cortactin. (A) Schematic diagram of BPGAP1 PP mutant, indicating substitution of the proline residues at 184 and 186 with alanines (arrows). (B) GST-recombinants of BPGAP1 FL and point mutant (PP) were used for pulldown assays by using lysates prepared from HeLa cells. Beads were washed and processed for Western analysis by using cortactin antibody. (C) Purified SH3(2) fusion of maltose binding protein, MBP-SH3(2), or the MBP control were incubated with Sepharose beads conjugated with purified GST recombinant proteins of either the full-length wild type or PP mutant of BPGAP1 or GST control, and bound targets revealed by Western blot analyses by using MBP antibody. Purified MBP and MBP-SH3(2) were analyzed with MBP antibody and revealed intact targets used in the direct binding assays. The lower apparent molecular weight for MBP-SH3(2) compared with the MBP alone was due to the removal of internal lacZ coding sequence upon cloning of the target insert. Blot was stripped and stained by amido black to verify loading of equal amounts of GSTs. (D) Cells were transfected with either the wild-type FL or PP mutant of FLAG-tagged BPGAP1 and immunoprecipitated with anti-FLAG M2 beads, washed, and analyzed for bound endogenous cortactin by using cortactin antibody. WCL, whole-cell lysates.

    Techniques Used: Binding Assay, Mutagenesis, Western Blot, Purification, Incubation, Recombinant, Molecular Weight, Sequencing, Clone Assay, Staining, Transfection, Immunoprecipitation

    31) Product Images from "Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit"

    Article Title: Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004938

    Bub2 GAP activity involves a ‘dual finger’ mechanism and promotes Bub2/Bfa1 clearance from the mother SPB. A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see Materials and Methods ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.
    Figure Legend Snippet: Bub2 GAP activity involves a ‘dual finger’ mechanism and promotes Bub2/Bfa1 clearance from the mother SPB. A-C: Bacterially purified GST-Bub2 or GST-Bub2-Q132L, MBP-Bfa1 and 6xHis-Tem1 proteins were used to measure the kinetics of hydrolysis+dissociation (γ[ 32 P]GTP) or dissociation only (γ[ 35 S]GTP) using a filter binding assay (see Materials and Methods ). Graphs show average values and standard deviations from three independent experiments. D: Exponentially growing cultures of the indicated strains were shifted to nocodazole containing medium at t = 0. Cell samples were withdrawn at the indicated time for FACS analysis of DNA contents. E: The percentage of cells with binucleate cell bodies accompanied or not by a checkpoint defect (indicated by re-budding in the absence of proper chromosome segregation) was scored in cycling cultures of the indicated strains shifted either to 14°C for 16h (left graph) or to 37°C for 3h (right graph). F-G: Exponentially growing cells with the indicated genotypes were arrested in G1 by α-factor and released into fresh medium at time 0. At 70’ after release α-factor was re-added to prevent cells from entering a second cell cycle. Cell samples were collected for FACS analysis of DNA contents (F) and for tubulin staining by indirect immunofluorescence (G). H: Cells were treated as in (F-G). TCA extracts were prepared from cell samples at the indicated time points to monitor kinetics of Bfa1-HA6 phosphorylation and Clb2 accumulation and degradation by western blot analysis. Pgk1 was used as loading control. I: Protein extracts from cells expressing the indicated tagged proteins were used for immunoprecipitation with an anti-HA affinity resin. Western blot analysis was then performed with anti-GFP and anti-HA antibodies. The input represents 1/25 th of the total extract used for each IP. J-K: Localization of eGFP- tagged Bub2/Bub2-Q132L, Tem1, Bfa1 (J) and Cdc15-GFP (K) was analysed by fluorescence microscopy after formaldehyde fixation.

    Techniques Used: Activity Assay, Purification, Filter-binding Assay, FACS, Staining, Immunofluorescence, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Microscopy

    The constitutively active Tem1-Q79L variant is checkpoint-deficient. A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in Material and Methods ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.
    Figure Legend Snippet: The constitutively active Tem1-Q79L variant is checkpoint-deficient. A: Bacterially purified 6XHis-Tem1 and 6XHis-Tem1-Q79L were loaded with γ[ 32 P]GTP either in the absence or in the presence of recombinant MBP-Bfa1 and incubated at 30°C for 10 minutes. The mixture was then added to GST-Bub2 or buffer alone and kinetics of GTP hydrolysis and dissociation was followed by a filter-binding assay (see details in Material and Methods ). Graphs show average values and standard deviations from three independent experiments. B: Wild type and TEM1-Q79L cells were arrested in G1 by α-factor and then released into fresh medium at 25°C (t = 0). Cell samples were withdrawn every 10’ to measure kinetics of budding and spindle formation/elongation after in situ immunostaining of tubulin. C: Actomyosin ring contraction has been visualized by live cell imaging of wild type and TEM1-Q79L expressing Myo1-GFP (n = 30). D: Logarithmically growing cultures of cells with the indicated genotypes were shifted into nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. E: The percentage of cells with binucleate cell bodies accompanied or not by SPOC defect was scored after DAPI staining of cycling cells of the indicated strains shifted to 14°C for 16h. F: Logarithmically growing cultures of strains with the indicated genotypes were shifted to nocodazole containing medium (t = 0). DNA contents were analysed by flow cytometry at the indicated times. G: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YPD or YP galactose plates and incubated at 30°C for 48h.

    Techniques Used: Variant Assay, Purification, Recombinant, Incubation, Filter-binding Assay, In Situ, Immunostaining, Live Cell Imaging, Expressing, Flow Cytometry, Cytometry, Staining

    32) Product Images from "Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181"

    Article Title: Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00876-15

    Bcl10 is a substrate for RNF181 E3 ubiquitin ligase activity in vitro . (A) Recombinant GST-RNF181 was incubated in in vitro ubiquitinylation reactions with E1, the indicated E2 enzymes, and either recombinant MBP-Bcl10 or MBP, as indicated. The products
    Figure Legend Snippet: Bcl10 is a substrate for RNF181 E3 ubiquitin ligase activity in vitro . (A) Recombinant GST-RNF181 was incubated in in vitro ubiquitinylation reactions with E1, the indicated E2 enzymes, and either recombinant MBP-Bcl10 or MBP, as indicated. The products

    Techniques Used: Activity Assay, In Vitro, Recombinant, Incubation

    33) Product Images from "Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains"

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Journal: PeerJ

    doi: 10.7717/peerj.3512

    Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.
    Figure Legend Snippet: Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Techniques Used: Western Blot, Size-exclusion Chromatography, Construct

    Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.
    Figure Legend Snippet: Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Techniques Used: Western Blot, Expressing, Construct

    34) Product Images from "Involvement of Girdin in the Determination of Cell Polarity during Cell Migration"

    Article Title: Involvement of Girdin in the Determination of Cell Polarity during Cell Migration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036681

    Identifying the Par-3 domain responsible for the interaction with Girdin. A. Fragments of the Par-3 4N domain used in the study are shown. The 4N domain of Par-3 was further divided into three subdomains (4N/1, 4N/2, and 4N/3). The corresponding amino acid numbers for the fragments are indicated in parenthesis. B. HEK293T cells were transfected with the indicated combination of each subdomain of myc-Par-3 4N, GST, and GST-CT. Lysates were precipitated with glutathione-Sepharose beads and eluted proteins were analysed with the indicated antibodies. Par-3 4N and 4N/2 domains that bound to GST-CT are indicated by asterisks. C. Direct interaction between the 4N/2 region of Par-3 and Girdin. Purified recombinant MBP (maltose binding protein)-fusion proteins containing the 4N, 4N/2, and 4N/1 regions of Par-3 (300 pmol) were incubated with 50 pmol of recombinant GST of GST-CT conjugated with glutathione beads for 1 hr at 4°C, washed three times, eluted with 10 mM reduced glutathione, separated on SDS-polyacrylamide gels, and subjected to Western blot analyses using the indicated antibodies. Asterisks, bound MBP-fusion proteins. White asterisks, MBP-fusion protein input. D. Total cell lysates from HEK293T cells that expressed Girdin-V5 and either myc-GST, myc-Par-3, or myc-Par-3 Δ4N/2 were immunoprecipitated with anti-myc antibody, followed by Western blot analyses using the indicated antibodies. myc-Par-3 (wild type and Δ4N/2) and Girdin-V5 are indicated by asterisks and stars, respectively.
    Figure Legend Snippet: Identifying the Par-3 domain responsible for the interaction with Girdin. A. Fragments of the Par-3 4N domain used in the study are shown. The 4N domain of Par-3 was further divided into three subdomains (4N/1, 4N/2, and 4N/3). The corresponding amino acid numbers for the fragments are indicated in parenthesis. B. HEK293T cells were transfected with the indicated combination of each subdomain of myc-Par-3 4N, GST, and GST-CT. Lysates were precipitated with glutathione-Sepharose beads and eluted proteins were analysed with the indicated antibodies. Par-3 4N and 4N/2 domains that bound to GST-CT are indicated by asterisks. C. Direct interaction between the 4N/2 region of Par-3 and Girdin. Purified recombinant MBP (maltose binding protein)-fusion proteins containing the 4N, 4N/2, and 4N/1 regions of Par-3 (300 pmol) were incubated with 50 pmol of recombinant GST of GST-CT conjugated with glutathione beads for 1 hr at 4°C, washed three times, eluted with 10 mM reduced glutathione, separated on SDS-polyacrylamide gels, and subjected to Western blot analyses using the indicated antibodies. Asterisks, bound MBP-fusion proteins. White asterisks, MBP-fusion protein input. D. Total cell lysates from HEK293T cells that expressed Girdin-V5 and either myc-GST, myc-Par-3, or myc-Par-3 Δ4N/2 were immunoprecipitated with anti-myc antibody, followed by Western blot analyses using the indicated antibodies. myc-Par-3 (wild type and Δ4N/2) and Girdin-V5 are indicated by asterisks and stars, respectively.

    Techniques Used: Transfection, Purification, Recombinant, Binding Assay, Incubation, Western Blot, Immunoprecipitation

    35) Product Images from "Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1"

    Article Title: Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038822

    Addition of phospholipids mixture reduces the STNB binding to GST-C2B. Binding of MBP STNA binding to GST-C2B is not affected by phospholipids (n=3). B. Phospholipids mixture containing phosphatidylserine (20% PE: phosphatidylethanolamine; 60% PC: phosphatidylcholine, 20% PS: phosphatidylserine) reduces the binding of MBP-STNB (n=4) to GST-C2B in comparison with binding in the presence of Ca2 + (*n=4, p
    Figure Legend Snippet: Addition of phospholipids mixture reduces the STNB binding to GST-C2B. Binding of MBP STNA binding to GST-C2B is not affected by phospholipids (n=3). B. Phospholipids mixture containing phosphatidylserine (20% PE: phosphatidylethanolamine; 60% PC: phosphatidylcholine, 20% PS: phosphatidylserine) reduces the binding of MBP-STNB (n=4) to GST-C2B in comparison with binding in the presence of Ca2 + (*n=4, p

    Techniques Used: Binding Assay

    The effect of pC2B1 peptide on STNA and STNB proteins binding and C2B self-association. The experiments were done in the presence of 1mM CaCl 2 . A. Addition of 10 µM pC2B1 reduced the MBP-STNA and abolished MBP-STNB binding to GST-C2B domain. B. Increasing concentration of pC2B1 in a dose dependent manner (0 to 4 µM) outcompeted MBP-STNB (0.25 µM) binding to 1 µM GST-C2B. The graph represents the amount of quantified MBP-STNA and MBP-STNB proteins normalized against the amount of eluted GST-C2B and the binding of MBP-Stoned in 0 µM pC2B1. Each value represents mean ± S.E.M (n=3). C. The pC2B1 peptide also reduced C2B SYT-1 oligomerization. The graph represents the amount of quantified MBP-C2B normalized against the amount of eluted GST- C2B. Each value represents mean ± S.E.M (n=3). The upper panel of each Western Blot image shows the MBP tagged proteins bound to GST-C2B and detected by anti-MBP antibody; while the lower panel is the Ponceau Red staining of GST proteins of the respective blots as loading control.
    Figure Legend Snippet: The effect of pC2B1 peptide on STNA and STNB proteins binding and C2B self-association. The experiments were done in the presence of 1mM CaCl 2 . A. Addition of 10 µM pC2B1 reduced the MBP-STNA and abolished MBP-STNB binding to GST-C2B domain. B. Increasing concentration of pC2B1 in a dose dependent manner (0 to 4 µM) outcompeted MBP-STNB (0.25 µM) binding to 1 µM GST-C2B. The graph represents the amount of quantified MBP-STNA and MBP-STNB proteins normalized against the amount of eluted GST-C2B and the binding of MBP-Stoned in 0 µM pC2B1. Each value represents mean ± S.E.M (n=3). C. The pC2B1 peptide also reduced C2B SYT-1 oligomerization. The graph represents the amount of quantified MBP-C2B normalized against the amount of eluted GST- C2B. Each value represents mean ± S.E.M (n=3). The upper panel of each Western Blot image shows the MBP tagged proteins bound to GST-C2B and detected by anti-MBP antibody; while the lower panel is the Ponceau Red staining of GST proteins of the respective blots as loading control.

    Techniques Used: Binding Assay, Concentration Assay, Western Blot, Staining

    Ca 2 dependent oligomerization regulates STNA and STNB proteins binding to the C2B domain of SYT-1. A. Truncation of C2B affects oligomerization. The N-terminal polylysine deletion (GST-ΔK) reduced MBP-C2B binding while the C-terminal tail deletion (GST-ΔW) increased MBP-C2B binding fourfold (n=3) in comparison with binding to GST-C2B. Independent experiments were done in Ca 2+ buffer. The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-ΔK or GST-ΔW is normalized against MBP-C2B bound to GST-C2B. Each value represents mean ± S.E.M. B. The D3,4N mutation abolished the effect of Ca 2 to regulate C2B oligomerization in comparison to GST-C2B in Ca 2 buffer (n=3, p
    Figure Legend Snippet: Ca 2 dependent oligomerization regulates STNA and STNB proteins binding to the C2B domain of SYT-1. A. Truncation of C2B affects oligomerization. The N-terminal polylysine deletion (GST-ΔK) reduced MBP-C2B binding while the C-terminal tail deletion (GST-ΔW) increased MBP-C2B binding fourfold (n=3) in comparison with binding to GST-C2B. Independent experiments were done in Ca 2+ buffer. The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-ΔK or GST-ΔW is normalized against MBP-C2B bound to GST-C2B. Each value represents mean ± S.E.M. B. The D3,4N mutation abolished the effect of Ca 2 to regulate C2B oligomerization in comparison to GST-C2B in Ca 2 buffer (n=3, p

    Techniques Used: Binding Assay, Mutagenesis

    Truncations of the C2B domain of SYT-1 affected STNA and STNB proteins binding. A. Several deletion constructs of C2B were created in respect of important residues known to alter the SYT-1 properties. B. Amino acid sequence of C-terminal truncated constructs of SYT-1-C2B domain. C. Deletion of the N-terminal polylysine region (GST-ΔK) reduced MBP-STNA and MBP-STNB proteins binding, in comparison with binding to GST-C2B. The graph represents the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST-C2B or GST-ΔK fusion proteins in Ca 2+ buffer. The amount of bound MBP-Stoned proteins to GST-ΔK is normalized against the binding to GST-C2B. Each value represents mean ± S.E.M. D. Deletions of C-terminal C2B increased the MBP-STNA and MBP-STNB binding. The graphs represent the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST fusion proteins in Ca 2+ buffer. The amount of bound MBP-Stoned proteins to GST-ΔH and GST-ΔW is normalized against MBP-Stoned proteins bound to GST-C2B. Experiments were done in Ca 2+ buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red stained of GST proteins of the respective blots as loading control.
    Figure Legend Snippet: Truncations of the C2B domain of SYT-1 affected STNA and STNB proteins binding. A. Several deletion constructs of C2B were created in respect of important residues known to alter the SYT-1 properties. B. Amino acid sequence of C-terminal truncated constructs of SYT-1-C2B domain. C. Deletion of the N-terminal polylysine region (GST-ΔK) reduced MBP-STNA and MBP-STNB proteins binding, in comparison with binding to GST-C2B. The graph represents the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST-C2B or GST-ΔK fusion proteins in Ca 2+ buffer. The amount of bound MBP-Stoned proteins to GST-ΔK is normalized against the binding to GST-C2B. Each value represents mean ± S.E.M. D. Deletions of C-terminal C2B increased the MBP-STNA and MBP-STNB binding. The graphs represent the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST fusion proteins in Ca 2+ buffer. The amount of bound MBP-Stoned proteins to GST-ΔH and GST-ΔW is normalized against MBP-Stoned proteins bound to GST-C2B. Experiments were done in Ca 2+ buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red stained of GST proteins of the respective blots as loading control.

    Techniques Used: Binding Assay, Construct, Sequencing, Western Blot, Staining

    Ca 2+ enhances the binding of the STNA and STNB proteins to the C2B domain of SYT-1. Preincubation of nucleic acid free GST-C2B with 1mM CaCl 2 in Hepes binding buffer increased the MBP-STNB (n=6) and MBP-STNA (n=6) binding, compared to preincubation in 1 mM EGTA containing buffer. In control, neither MBP-STNA nor MBP-STNB binds to GST protein in the presence of 1 mM CaCl 2 . The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in Ca 2+ are normalized against MBP-Stoned proteins bound in EGTA buffer. Each value represents mean ± S.E.M. B. The STNA and STNB proteins, bound to GST-C2B in the presence of Ca 2+ , were subsequently washed with 1 mM EGTA-containing buffer. Washing in EGTA reduced the amount of bound MBP-STNB (n=3) and MBP-STNA (n=3) proteins, compared with equivalent washes in buffer containing 1 mM CaCl 2 (Ca 2+ washing buffer). The bar graph represents the amount of quantified MBP-Stoned proteins. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in EGTA buffer is normalized against MBP-Stoned proteins bound to GST-C2B in Ca 2+ buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B of the respective blots as loading control.
    Figure Legend Snippet: Ca 2+ enhances the binding of the STNA and STNB proteins to the C2B domain of SYT-1. Preincubation of nucleic acid free GST-C2B with 1mM CaCl 2 in Hepes binding buffer increased the MBP-STNB (n=6) and MBP-STNA (n=6) binding, compared to preincubation in 1 mM EGTA containing buffer. In control, neither MBP-STNA nor MBP-STNB binds to GST protein in the presence of 1 mM CaCl 2 . The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in Ca 2+ are normalized against MBP-Stoned proteins bound in EGTA buffer. Each value represents mean ± S.E.M. B. The STNA and STNB proteins, bound to GST-C2B in the presence of Ca 2+ , were subsequently washed with 1 mM EGTA-containing buffer. Washing in EGTA reduced the amount of bound MBP-STNB (n=3) and MBP-STNA (n=3) proteins, compared with equivalent washes in buffer containing 1 mM CaCl 2 (Ca 2+ washing buffer). The bar graph represents the amount of quantified MBP-Stoned proteins. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in EGTA buffer is normalized against MBP-Stoned proteins bound to GST-C2B in Ca 2+ buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B of the respective blots as loading control.

    Techniques Used: Binding Assay, Western Blot, Staining

    36) Product Images from "An ExbD Disordered Domain Peptide Inhibits TonB System Activity"

    Article Title: An ExbD Disordered Domain Peptide Inhibits TonB System Activity

    Journal: bioRxiv

    doi: 10.1101/2020.01.05.895219

    dsbA(ss)-ExbD(44-63; V45A, V47A) and dsbA(ss)-ExbD(49-63) do not inhibit the ExbD-TonB complex and reduced TonB interactions. Formaldehyde cross-linking of W3110 (WT) expressing the dsbA(ss)-ExbD(44-63), dsbA(ss)-ExbD(44-63; V45A, V47A) or dsbA(ss)-ExbD(49-63) probed with (A) anti-ExbD polyclonal antibodies or (B) anti-TonB monoclonal antibodies. Strains were grown to mid-exponential phase, at which point formaldehyde-cross-linking was performed as described in Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 13% SDS-polyacrylamide gels. (A, top) (lane 1) WT + pBAD33 refers to W3110 harboring the empty vector pBAD33 ExbD(44-63), (lane 2) ExbD(44-63), (lane 3) ExbD(44-63; V45A, V47A), and (lane 4) ExbD(49-63) refer to W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63), dsbA(ss)-ExbD(44-63; V45A, V47A), and dsbA(ss)-ExbD(49-63). (A, right) Positions of the previously characterized ExbD formaldehyde cross-linked complexes are shown: the PMF-dependent ExbD-TonB complex (red), the ExbD-ExbB complex, the ExbD homodimer (ExbD-ExbD), and ExbD monomer ( 2 ). (right, labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with ExbD. (right in green) identifies the 17.1 kDa unknown ExbD complex (A, right) Mass markers are shown. (A, bottom) A shorter exposure of ExbD monomer corresponding to each sample. (B, top) (lane 1) WT + pBAD33 refers to W3110 harboring the empty vector pBAD33, (lane 2) ExbD(44-63), (lane 3) ExbD(44-63; V45A, V47A), and (lane 4) ExbD(49-63) refer to W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X, dsbA(ss)-ExbD(44-63; V45A, V47A)-His6X, and dsbA(ss)-ExbD(49-63)-His6X. (B, right) Positions of the previously characterized TonB formaldehyde cross-linked complexes are shown TonB-FepA complex, the TonB-ExbB complex, the PMF-dependent TonB-ExbD complex (red), TonB-LPP (Braun’s lipoprotein) and TonB monomer (3, 2). (B, right, labeled in purple) The suspected complexes of the peptide trapped with TonB: TonB-peptide1A, TonB-peptide1B, and TonB-peptide1C. (B, left) Mass markers are shown. (B, bottom) A shorter exposure of TonB monomer corresponding to each sample. (C) Relative expression of plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X, dsbA(ss)-ExbD(44-63; V45A, V47A)-His6X, and dsbA(ss)-ExbD(49-63)-His6X in in whole cells (WC) and in the periplasm (Peri). Strains were grown to mid-exponential phase, at which point whole cells and soluble periplasmic samples were collected as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. Protein expression was analyzed by probing with (Top panel) anti-maltose-binding protein (MBP) antibodies and (Bottom panel) anti-His monoclonal antibodies. The MBP, peptides, WC, periplasmic fractions were all from the same gel and the same immunoblot exposure for relative comparison. The plasmid identities are listed in Table S1.
    Figure Legend Snippet: dsbA(ss)-ExbD(44-63; V45A, V47A) and dsbA(ss)-ExbD(49-63) do not inhibit the ExbD-TonB complex and reduced TonB interactions. Formaldehyde cross-linking of W3110 (WT) expressing the dsbA(ss)-ExbD(44-63), dsbA(ss)-ExbD(44-63; V45A, V47A) or dsbA(ss)-ExbD(49-63) probed with (A) anti-ExbD polyclonal antibodies or (B) anti-TonB monoclonal antibodies. Strains were grown to mid-exponential phase, at which point formaldehyde-cross-linking was performed as described in Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 13% SDS-polyacrylamide gels. (A, top) (lane 1) WT + pBAD33 refers to W3110 harboring the empty vector pBAD33 ExbD(44-63), (lane 2) ExbD(44-63), (lane 3) ExbD(44-63; V45A, V47A), and (lane 4) ExbD(49-63) refer to W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63), dsbA(ss)-ExbD(44-63; V45A, V47A), and dsbA(ss)-ExbD(49-63). (A, right) Positions of the previously characterized ExbD formaldehyde cross-linked complexes are shown: the PMF-dependent ExbD-TonB complex (red), the ExbD-ExbB complex, the ExbD homodimer (ExbD-ExbD), and ExbD monomer ( 2 ). (right, labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with ExbD. (right in green) identifies the 17.1 kDa unknown ExbD complex (A, right) Mass markers are shown. (A, bottom) A shorter exposure of ExbD monomer corresponding to each sample. (B, top) (lane 1) WT + pBAD33 refers to W3110 harboring the empty vector pBAD33, (lane 2) ExbD(44-63), (lane 3) ExbD(44-63; V45A, V47A), and (lane 4) ExbD(49-63) refer to W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X, dsbA(ss)-ExbD(44-63; V45A, V47A)-His6X, and dsbA(ss)-ExbD(49-63)-His6X. (B, right) Positions of the previously characterized TonB formaldehyde cross-linked complexes are shown TonB-FepA complex, the TonB-ExbB complex, the PMF-dependent TonB-ExbD complex (red), TonB-LPP (Braun’s lipoprotein) and TonB monomer (3, 2). (B, right, labeled in purple) The suspected complexes of the peptide trapped with TonB: TonB-peptide1A, TonB-peptide1B, and TonB-peptide1C. (B, left) Mass markers are shown. (B, bottom) A shorter exposure of TonB monomer corresponding to each sample. (C) Relative expression of plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X, dsbA(ss)-ExbD(44-63; V45A, V47A)-His6X, and dsbA(ss)-ExbD(49-63)-His6X in in whole cells (WC) and in the periplasm (Peri). Strains were grown to mid-exponential phase, at which point whole cells and soluble periplasmic samples were collected as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. Protein expression was analyzed by probing with (Top panel) anti-maltose-binding protein (MBP) antibodies and (Bottom panel) anti-His monoclonal antibodies. The MBP, peptides, WC, periplasmic fractions were all from the same gel and the same immunoblot exposure for relative comparison. The plasmid identities are listed in Table S1.

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Peptide Mass Fingerprinting, Labeling, Binding Assay

    dsbA(ss)-ExbD(44-63) is inefficiently localized to the periplasm. Proteinase K (PK) accessibility in Whole cells (WC), Spheroplasts (Sph), Lysed Spheroplasts (Lys), and periplasmic fraction (Peri) of W3110 expressing plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X (pKP1960). Strains were grown to an A 550 of 0.2 at which point 0.2% (w/v) of arabinose was added to induce intracellular expression of the dsbA(ss)-ExbD(44-63)-His6X. Strains were grown until mid-exponential phase. WC, Sph, Lys, and Peri fractions treated (+) and untreated (-) with Proteinase K as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. The periplasmic soluble maltose-binding protein (MBP) was probed with anti-maltose-binding protein monoclonal antibodies (Top panel). The cytoplasmic membrane-embedded, periplasmic TonB protein was probed with anti-TonB monoclonal antibodies (Bottom panel) and ExbD(44-63)-His peptide was probed with anti-His monoclonal antibodies. The MBP, TonB, and ExbD(44-63)-His were all probed from same immunoblot.
    Figure Legend Snippet: dsbA(ss)-ExbD(44-63) is inefficiently localized to the periplasm. Proteinase K (PK) accessibility in Whole cells (WC), Spheroplasts (Sph), Lysed Spheroplasts (Lys), and periplasmic fraction (Peri) of W3110 expressing plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X (pKP1960). Strains were grown to an A 550 of 0.2 at which point 0.2% (w/v) of arabinose was added to induce intracellular expression of the dsbA(ss)-ExbD(44-63)-His6X. Strains were grown until mid-exponential phase. WC, Sph, Lys, and Peri fractions treated (+) and untreated (-) with Proteinase K as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. The periplasmic soluble maltose-binding protein (MBP) was probed with anti-maltose-binding protein monoclonal antibodies (Top panel). The cytoplasmic membrane-embedded, periplasmic TonB protein was probed with anti-TonB monoclonal antibodies (Bottom panel) and ExbD(44-63)-His peptide was probed with anti-His monoclonal antibodies. The MBP, TonB, and ExbD(44-63)-His were all probed from same immunoblot.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Binding Assay

    dsbA(ss)-ExbD(44-63) depends on the presence of ExbD and TonB to interact with TonB and ExbD respectively, but not PMF. Formaldehyde cross-linking of W3110 (WT) with a plasmid expressing of dsbA(ss)-ExbD(44-63) in the absence of PMF, and in the absence of the exbD gene, and in the absence of the tonB gene. Samples were probed with (A) anti-ExbD polyclonal antibodies or (B) anti-TonB monoclonal antibodies. Strains were grown to mid-exponential phase, at which point formaldehyde-cross-linking was performed as described in Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 13% SDS-polyacrylamide gels. (lane 1) ΔexbD, ΔtolQR refers to W3110 with the exbD gene and its homolog genes tolQR deleted (RA1045). (lane 2) ΔtonB refers to W3110 with the tonB gene deleted (KP1477). WT + pBAD33 refers to W3110 harboring the empty vector pBAD33 without (lane 3) and with (lane 4) the 60µM CCCP. WT + ExbD(44-63) is W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose without (lane 4) and with (lane 5) the 60µM CCCP. (lane 6) ΔexbD, ΔtolQR + ExbD(44-63) refers to RA1045 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose. (lane 7) ΔtonB + ExbD(44-63) refers to KP1477 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose. (A, right) Positions of the previously characterized ExbD formaldehyde cross-linked complexes are shown: the PMF-dependent ExbD-TonB complex (red), the ExbD-ExbB complex, the ExbD homodimer (ExbD-ExbD), and ExbD monomer ( 25 ). (A, right labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with ExbD. (A, left) Mass markers are shown. (A, below) A shorter exposure of ExbD monomer corresponding to each sample. (B, right) Positions of the previously characterized TonB formaldehyde cross-linked complexes are shown TonB-FepA complex, the TonB-ExbB complex, the PMF-dependent TonB-ExbD complex, TonB-LPP (Braun’s lipoprotein) and TonB monomer (35, 25). (B, right labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with TonB. (left) Mass markers are shown. (B, below) A shorter exposure of TonB monomer corresponding to each sample. (C) Relative expression of plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X expressed in W3110, KP1477, and RA1045 in whole cells (WC) and in the periplasm (Peri). Strains were grown to mid-exponential phase, at which point whole cells and soluble periplasmic samples were collected as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. Protein expression was analyzed by probing with (Top panel) anti-maltose-binding protein (MBP) antibodies and (Bottom panel) anti-His monoclonal antibodies. The MBP, peptides, WC, periplasmic fractions were all from the same gel and the same immunoblot exposure for relative comparison. The plasmid identities are listed in Table S1.
    Figure Legend Snippet: dsbA(ss)-ExbD(44-63) depends on the presence of ExbD and TonB to interact with TonB and ExbD respectively, but not PMF. Formaldehyde cross-linking of W3110 (WT) with a plasmid expressing of dsbA(ss)-ExbD(44-63) in the absence of PMF, and in the absence of the exbD gene, and in the absence of the tonB gene. Samples were probed with (A) anti-ExbD polyclonal antibodies or (B) anti-TonB monoclonal antibodies. Strains were grown to mid-exponential phase, at which point formaldehyde-cross-linking was performed as described in Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 13% SDS-polyacrylamide gels. (lane 1) ΔexbD, ΔtolQR refers to W3110 with the exbD gene and its homolog genes tolQR deleted (RA1045). (lane 2) ΔtonB refers to W3110 with the tonB gene deleted (KP1477). WT + pBAD33 refers to W3110 harboring the empty vector pBAD33 without (lane 3) and with (lane 4) the 60µM CCCP. WT + ExbD(44-63) is W3110 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose without (lane 4) and with (lane 5) the 60µM CCCP. (lane 6) ΔexbD, ΔtolQR + ExbD(44-63) refers to RA1045 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose. (lane 7) ΔtonB + ExbD(44-63) refers to KP1477 expressing the plasmid-encoded dsbA(ss)-ExbD(44-63) under the arabinose inducible promoter (pKP1847) with 0.2% (w/v) arabinose. (A, right) Positions of the previously characterized ExbD formaldehyde cross-linked complexes are shown: the PMF-dependent ExbD-TonB complex (red), the ExbD-ExbB complex, the ExbD homodimer (ExbD-ExbD), and ExbD monomer ( 25 ). (A, right labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with ExbD. (A, left) Mass markers are shown. (A, below) A shorter exposure of ExbD monomer corresponding to each sample. (B, right) Positions of the previously characterized TonB formaldehyde cross-linked complexes are shown TonB-FepA complex, the TonB-ExbB complex, the PMF-dependent TonB-ExbD complex, TonB-LPP (Braun’s lipoprotein) and TonB monomer (35, 25). (B, right labeled in purple) The suspected complexes of the dsbA(ss)-ExbD(44-63) peptide trapped with TonB. (left) Mass markers are shown. (B, below) A shorter exposure of TonB monomer corresponding to each sample. (C) Relative expression of plasmid-encoded dsbA(ss)-ExbD(44-63)-His6X expressed in W3110, KP1477, and RA1045 in whole cells (WC) and in the periplasm (Peri). Strains were grown to mid-exponential phase, at which point whole cells and soluble periplasmic samples were collected as described in the Materials and Methods. Equivalent numbers of cells were visualized on immunoblots of 16% acrylamide/ 6% bis-acrylamide SDS tricine gels. Protein expression was analyzed by probing with (Top panel) anti-maltose-binding protein (MBP) antibodies and (Bottom panel) anti-His monoclonal antibodies. The MBP, peptides, WC, periplasmic fractions were all from the same gel and the same immunoblot exposure for relative comparison. The plasmid identities are listed in Table S1.

    Techniques Used: Peptide Mass Fingerprinting, Plasmid Preparation, Expressing, Western Blot, Labeling, Binding Assay

    37) Product Images from "SPAs promote thermomorphogenesis via regulating the phyB-PIF4 module in Arabidopsis"

    Article Title: SPAs promote thermomorphogenesis via regulating the phyB-PIF4 module in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/2020.02.07.938951

    Interaction between SPA1 and PIF4. (A) yeast two hybrid of SPA1 and PIF4. LexA-PIF4 was co-transformed with empty AD or AD-SPA1. The error bars represent standard deviation. Three biological replicates were used in this study. (B) in vitro pull-down assay using MBP-SPA1 and GST-PIF4. GST only was used for negative control. Single asterisk mark (*) shows GST-PIF4 band and double asterisk mark (**) shows GST only protein.
    Figure Legend Snippet: Interaction between SPA1 and PIF4. (A) yeast two hybrid of SPA1 and PIF4. LexA-PIF4 was co-transformed with empty AD or AD-SPA1. The error bars represent standard deviation. Three biological replicates were used in this study. (B) in vitro pull-down assay using MBP-SPA1 and GST-PIF4. GST only was used for negative control. Single asterisk mark (*) shows GST-PIF4 band and double asterisk mark (**) shows GST only protein.

    Techniques Used: Transformation Assay, Standard Deviation, In Vitro, Pull Down Assay, Negative Control

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    New England Biolabs anti mbp monoclonal antibody
    Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in <t>V5-tagged</t> Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding <t>protein-(MS2-MBP)-conjugated</t> agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.
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    Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in V5-tagged Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding protein-(MS2-MBP)-conjugated agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling

    doi: 10.1038/s41467-021-23459-4

    Figure Lengend Snippet: Prp28 contacts key proteins at the heart of spliceosome. a Low ATP traps a transient interaction of Prp28 with the spliceosome. Splicing reactions (lanes 1–5) were done in V5-tagged Prp28 extracts at 0, 0.02, 0.05, 0.2, or 2 mM ATP and a portion was subjected to immunoprecipitation without antibody (PAS; lanes 6–10) or with anti-V5 antibody (lanes 11–15). Relative loadings are 1:10 for splicing reactions alone (lanes 1–5) vs. immunoprecipitated reactions (lanes 6–15). Positions of pre-mRNA, splicing intermediates, and mRNA are indicated to the left. The experiment was repeated three times with similar results. b Schematic diagram showing a BPA-marked Prp28 cross-linked to protein X in a spliceosome assembled on MS2 stem-loop-tagged ACT1 pre-mRNA, which can be pulled down by MS2-maltose-binding protein-(MS2-MBP)-conjugated agarose beads. Thunderbolt, 365-nm UV irradiation. c Prp28-BPA cross-linked species (Prp28-X) detected by using anti-Prp28, or using anti-HA and anti-V5 tag antibody for Prp28-tagged experiments. K27, K41, K82, and K136 are the amino-acid residues in Prp28 replaced by BPA. (−) and (+), without or with UV irradiation, respectively. Filled circle, uncrosslinked Prp28. Asterisk, nonspecific background band. Detection of MS2-MBP serves as a loading control. The experiments were repeated three times with similar results. d Identification of the X proteins as Prp8, Brr2, Snu114, and U1C by using anti-Prp8, anti-Brr2, anti-Snu114, or anti-V5 (U1C-V5) antibody, respectively. The experiments were repeated three times with similar results. e Schematic summary of the cross-linking data. Splicing complexes accumulated at various ATP concentrations are shown to the left. The changing amount of Prp28 associated with the spliceosome is depicted to the right. Source data are provided as a Source Data file.

    Article Snippet: In addition, the following reagents were from commercial sources: p -Benzoyl-l -phenylalanine (Bpa) (Bachem), anti-HA.11 (ms, Cat # MMS-101R, Lot # B220850, Covance, Clone: 16B12, 1:2000), anti-V5-TAG (ms, Cat # MCA1360, Lot # 0915, Bio-Rad, Clone: SV5-PK1, 1:2000), anti-Maltose Binding Protein (MBP) (ms, Cat # E8032S, Lot # 0101603, NEB, Clone: B48, 1:10,000), anti-GAPDH (ms, Cat # G8795-200, Lot # 045M4799V, Sigma, 1:10000), HRP-conjugated anti-rabbit IgG (H + L) (gt, Cat # 65–6120, Lot # QK229568, Invitrogen, 1:10,000–1:40,000), HRP-conjugated anti-mouse IgG (H+L) (gt, Cat # 62–6520, Lot # QG215721, Invitrogen, 1:10,000~1:40,000), Immobilon Western Chemiluminescent HRP Substrate (Millipore), SuperSignal West Femto Maximum Sensitivity Substrate (Thermo), Polyvinylidene difluoride membrane (AmershamTM Hybond 0.45 mm PVDF, GE), Protein A-Sepharose (PAS) was obtained from GE Healthcare Life Sciences, Protein A Mag Sepharose Xtra (PAmS) (GE), Amylose Resin (New England Biolabs), 5-Fluoroorotic acid (5-FOA) (ZYMO RESEARCH), Proteinase K was purchased from MD Bio Inc., and Fast SYBR Green Master Mix (Applied Biosystems), Perfect RNA marker template mix (0.1–1 kb, Novagen, Cat # 69003-3).

    Techniques: Immunoprecipitation, Binding Assay, Irradiation

    SDS-PAGE of MBP-VP4 fusion proteins. The samples of MBP fusion protein containing either wild-type or mutant forms of the leucine zipper region of VP4 were analyzed by SDS-PAGE (10% polyacrylamide). Lanes: 1, purified MBP; 2, purified fusion protein derived from plasmid MBP/VP4LZ expressing the wild-type leucine zipper region; 3 and 4, standard molecular mass markers; 5, purified fusion protein derived from plasmid MBP/VP4LZP expressing the mutant (L537P) leucine zipper region.

    Journal: Journal of Virology

    Article Title: A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4

    doi:

    Figure Lengend Snippet: SDS-PAGE of MBP-VP4 fusion proteins. The samples of MBP fusion protein containing either wild-type or mutant forms of the leucine zipper region of VP4 were analyzed by SDS-PAGE (10% polyacrylamide). Lanes: 1, purified MBP; 2, purified fusion protein derived from plasmid MBP/VP4LZ expressing the wild-type leucine zipper region; 3 and 4, standard molecular mass markers; 5, purified fusion protein derived from plasmid MBP/VP4LZP expressing the mutant (L537P) leucine zipper region.

    Article Snippet: The membranes were blocked with 5% fat-free milk powder in phosphate-buffered saline for 1 h and probed with a polyclonal guinea pig anti-VP4 antiserum (dilution of 1:1,000), an anti-MBP monoclonal anti-serum (NEB), or a polyclonal rabbit anti-BTV antiserum for 60 min at room temperature.

    Techniques: SDS Page, Mutagenesis, Purification, Derivative Assay, Plasmid Preparation, Expressing

    Dppa2 inhibits microtubule assembly around chromatin and in vitro (A) Dppa2 inhibits sperm aster microtubule assembly. Demembranated sperm nuclei were added together with calcium to metaphase extracts supplemented with rhodamine-labeled tubulin (red). Samples were fixed and stained with Hoechst 33342 (blue). (B) Quantification of tubulin fluorescence intensity from (A). Data shown indicate mean and standard error from > 30 asters per sample and are representative of 3 independent experiments. (C) Dppa2 inhibits spindle assembly in a dose-dependent manner. Metaphase spindles were assembled in extracts supplemented with MBP-Dppa2 fusion proteins and rhodamine-labeled tubulin. (D) Quantification of spindle length from (C). Data shown are mean and standard deviation from 30 spindles per sample. (E) Chromatin-independent microtubule assembly in Xenopus egg extracts. Recombinant GST-Dppa2 protein was added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (F) Dppa2 inhibits microtubule polymerization in vitro . MBP-Dppa2 was added to purified bovine tubulin together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. Scale bars, 10 μm. .

    Journal: Developmental cell

    Article Title: Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

    doi: 10.1016/j.devcel.2013.08.002

    Figure Lengend Snippet: Dppa2 inhibits microtubule assembly around chromatin and in vitro (A) Dppa2 inhibits sperm aster microtubule assembly. Demembranated sperm nuclei were added together with calcium to metaphase extracts supplemented with rhodamine-labeled tubulin (red). Samples were fixed and stained with Hoechst 33342 (blue). (B) Quantification of tubulin fluorescence intensity from (A). Data shown indicate mean and standard error from > 30 asters per sample and are representative of 3 independent experiments. (C) Dppa2 inhibits spindle assembly in a dose-dependent manner. Metaphase spindles were assembled in extracts supplemented with MBP-Dppa2 fusion proteins and rhodamine-labeled tubulin. (D) Quantification of spindle length from (C). Data shown are mean and standard deviation from 30 spindles per sample. (E) Chromatin-independent microtubule assembly in Xenopus egg extracts. Recombinant GST-Dppa2 protein was added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (F) Dppa2 inhibits microtubule polymerization in vitro . MBP-Dppa2 was added to purified bovine tubulin together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. Scale bars, 10 μm. .

    Article Snippet: Primary antibodies were diluted in AbDil (10 mM Tris, 150 mM NaCl, 2 % BSA, 0.1% Triton X-100, pH 7.4) as follows: 6 μg/ml anti-Dasra A ( ); 0.2 μg/ml anti-Dppa2; 2 μg/ml anti-GFP (Roche 11814460001); 1 μg/ml anti-H3S10ph (mAb 7G1G7, gift of Hiroshi Kimura); 1:200 anti-lamin B3 (gift of Dale K. Shumaker); 0.2 μg/ml mAb414 (Covance MMS-120P); 0.2 μg/ml anti-MCAK (gift of Ryoma Ohi); 1 μg/ml anti-MBP (NEB E8032), 1:1,000 anti-α-tubulin (Sigma T9026).

    Techniques: In Vitro, Labeling, Staining, Fluorescence, Standard Deviation, Recombinant, Purification

    Dppa2 requires its C terminus but not DNA-binding to inhibit microtubule assembly (A) Schematic of Dppa2 deletion constructs used. (B) Inhibition of spindle assembly requires the Dppa2 C terminus but not DNA-binding. Metaphase spindles were assembled in extracts supplemented with 2 μM MBP-Dppa2 fusion proteins. MBP-Dppa2 localization was visualized by immunofluorescence using an anti-MBP antibody. Scale bar, 10 μm. (C) Quantification of spindle length in (B). Bars represent mean and standard error from 30 spindles and are representative of 3 independent experiments. (D) Chromatin-independent microtubule assembly in Xenopus egg extracts. MBP-Dppa2 proteins were added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (E) Polymerization of purified tubulin in vitro . Purified bovine tubulin was treated with MBP-Dppa2 proteins and 0.5 % DMSO, and polymerized microtubules analyzed by Coomassie staining.

    Journal: Developmental cell

    Article Title: Chromatin-Bound Xenopus Dppa2 Shapes the Nucleus by Locally Inhibiting Microtubule Assembly

    doi: 10.1016/j.devcel.2013.08.002

    Figure Lengend Snippet: Dppa2 requires its C terminus but not DNA-binding to inhibit microtubule assembly (A) Schematic of Dppa2 deletion constructs used. (B) Inhibition of spindle assembly requires the Dppa2 C terminus but not DNA-binding. Metaphase spindles were assembled in extracts supplemented with 2 μM MBP-Dppa2 fusion proteins. MBP-Dppa2 localization was visualized by immunofluorescence using an anti-MBP antibody. Scale bar, 10 μm. (C) Quantification of spindle length in (B). Bars represent mean and standard error from 30 spindles and are representative of 3 independent experiments. (D) Chromatin-independent microtubule assembly in Xenopus egg extracts. MBP-Dppa2 proteins were added to metaphase extracts together with 0.5 % DMSO. Polymerized microtubules were recovered by pelleting and analyzed by Coomassie staining. (E) Polymerization of purified tubulin in vitro . Purified bovine tubulin was treated with MBP-Dppa2 proteins and 0.5 % DMSO, and polymerized microtubules analyzed by Coomassie staining.

    Article Snippet: Primary antibodies were diluted in AbDil (10 mM Tris, 150 mM NaCl, 2 % BSA, 0.1% Triton X-100, pH 7.4) as follows: 6 μg/ml anti-Dasra A ( ); 0.2 μg/ml anti-Dppa2; 2 μg/ml anti-GFP (Roche 11814460001); 1 μg/ml anti-H3S10ph (mAb 7G1G7, gift of Hiroshi Kimura); 1:200 anti-lamin B3 (gift of Dale K. Shumaker); 0.2 μg/ml mAb414 (Covance MMS-120P); 0.2 μg/ml anti-MCAK (gift of Ryoma Ohi); 1 μg/ml anti-MBP (NEB E8032), 1:1,000 anti-α-tubulin (Sigma T9026).

    Techniques: Binding Assay, Construct, Inhibition, Immunofluorescence, Staining, Purification, In Vitro

    Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Journal: bioRxiv

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    doi: 10.1101/113142

    Figure Lengend Snippet: Western blot of the first peak from SEC using anti-MBP antibody. Arrow points to band correlating to FGFR2 construct size (71.5 kDa). Lane 1: Ladder. Lane 2: SEC fraction A12. Both lanes are cropped from the same blot.

    Article Snippet: We were able to express FGFRs and avoid expression toxicity using E. coli strain Lemo21, that contains T7 RNA polymerase that is blot with an anti-MBP antibody showed significant quantities of FGFR2 and FGFR3 ECD + TM in both the soluble and cell pellet fractions ( , lanes 2, 4, 7, 9).

    Techniques: Western Blot, Construct

    Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Journal: bioRxiv

    Article Title: Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    doi: 10.1101/113142

    Figure Lengend Snippet: Western blot analysis of small-scale expression of FGFR2 and FGFR3 constructs using anti-MBP antibody. Lane 1: Ladder. Lane 2: FGFR2 31-406 from supernatant. Lane 3: FGFR2 370-651 from supernatant. Lane 4: FGFR3 143-405 from supernatant. Lane 5: FGFR 3: 365-771 from supernatant. Lane 6: Blank. Lane 7: FGFR2 31-406 from pellet. Lane 8: FGFR2 370-651 from pellet. Lane 9: FGFR3 143-405 from pellet. Lane 10: FGFR3 365-771 from pellet. Circled in red are bands consistent with FGFR2 and FGFR3 ECD + TM from supernatant. Boxed in blue are bands consistent with FGFR 2 and 3 ECD + TM from the cell pellet fraction. Boxed in green is a band consistent with FGFR3 TM + KD from the cell pellet fraction.

    Article Snippet: We were able to express FGFRs and avoid expression toxicity using E. coli strain Lemo21, that contains T7 RNA polymerase that is blot with an anti-MBP antibody showed significant quantities of FGFR2 and FGFR3 ECD + TM in both the soluble and cell pellet fractions ( , lanes 2, 4, 7, 9).

    Techniques: Western Blot, Expressing, Construct