mbp  (New England Biolabs)


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  • 99
    Name:
    Anti MBP Monoclonal Antibody
    Description:
    Anti MBP Monoclonal Antibody 0 25 ml
    Catalog Number:
    E8032L
    Price:
    698
    Size:
    0 25 ml
    Category:
    Primary Antibodies
    Score:
    85
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    Structured Review

    New England Biolabs mbp
    Anti MBP Monoclonal Antibody
    Anti MBP Monoclonal Antibody 0 25 ml
    https://www.bioz.com/result/mbp/product/New England Biolabs
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    mbp - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Sec16p potentiates the action of COPII proteins to bud transport vesicles"

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200207053

    Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.
    Figure Legend Snippet: Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.

    Techniques Used: SDS Page

    Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.
    Figure Legend Snippet: Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.

    Techniques Used: Incubation, SDS Page, Staining

    Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).
    Figure Legend Snippet: Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).

    Techniques Used: Incubation, Fluorescence, Labeling

    Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.
    Figure Legend Snippet: Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.

    Techniques Used: Binding Assay, Incubation, SDS Page, Staining, Titration

    Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.
    Figure Legend Snippet: Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.

    Techniques Used: Titration, Binding Assay, Quantitation Assay

    Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.
    Figure Legend Snippet: Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.

    Techniques Used: Over Expression, Purification, Incubation, SDS Page, Staining, Molecular Weight

    Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.
    Figure Legend Snippet: Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.

    Techniques Used: Electron Microscopy, Incubation

    2) Product Images from "Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells"

    Article Title: Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.221

    Identification of Fra-2 phospho-acceptor sites using phospho-peptide mass spectrometry analysis. ( a ) Purified GST, GST-Fra-2 or MBP were incubated with γ - 32 P-ATP and activated ERK 2 or its buffer in vitro . The samples were run on an 8% SDS-PAGE, which was Coomassie stained ( a ), dried and exposed to film ( b ). GST alone was used as a negative control and MBP was used as a positive control. ( b ) Linear Fra-2 amino acid sequence. The in vitro kinase assay was repeated with GST-Fra-2, activated ERK 2 and unlabeled ATP. Samples were resolved on an 8% SDS-PAGE, and the unphosphorylated and phosphorylated GST-Fra-2 bands were excised, digested by trypsin and the resultant peptides were analyzed by mass spectrometry. The results from the mass spectrometry analysis are summarized: where phosphopeptides detected by mass spectrometry are boxed, DEF domain (282-285) is underlined and ERK 2 phosphorylated residues (120, 200, 230, 320, 322) are in bold. ( c ) Spectra for S320 (upper panel). C13 ion has a m/z of 1269.7 and C15 ion has a m/z of 1534.8, and a difference of 265.1. The C13 ion is composed of S and P that have masses of 87.08 and 97.12, respectively, added together to give a value of 184.2. The difference between 265.1 and 184.2 is 80.90, which corresponds to a addition of a phosphate group. The spectra for T322 (lower panel) has a C13 of 1269.6 and C16 of 1634.7, and has a difference of 365.1. The C16 ion corresponds to S, P and T, which m/z of 87.08, 97.12, and 101.11, respectively, added together gives 285.31. The difference of the value gives 79.79, corresponding to an addition of a phosphate group
    Figure Legend Snippet: Identification of Fra-2 phospho-acceptor sites using phospho-peptide mass spectrometry analysis. ( a ) Purified GST, GST-Fra-2 or MBP were incubated with γ - 32 P-ATP and activated ERK 2 or its buffer in vitro . The samples were run on an 8% SDS-PAGE, which was Coomassie stained ( a ), dried and exposed to film ( b ). GST alone was used as a negative control and MBP was used as a positive control. ( b ) Linear Fra-2 amino acid sequence. The in vitro kinase assay was repeated with GST-Fra-2, activated ERK 2 and unlabeled ATP. Samples were resolved on an 8% SDS-PAGE, and the unphosphorylated and phosphorylated GST-Fra-2 bands were excised, digested by trypsin and the resultant peptides were analyzed by mass spectrometry. The results from the mass spectrometry analysis are summarized: where phosphopeptides detected by mass spectrometry are boxed, DEF domain (282-285) is underlined and ERK 2 phosphorylated residues (120, 200, 230, 320, 322) are in bold. ( c ) Spectra for S320 (upper panel). C13 ion has a m/z of 1269.7 and C15 ion has a m/z of 1534.8, and a difference of 265.1. The C13 ion is composed of S and P that have masses of 87.08 and 97.12, respectively, added together to give a value of 184.2. The difference between 265.1 and 184.2 is 80.90, which corresponds to a addition of a phosphate group. The spectra for T322 (lower panel) has a C13 of 1269.6 and C16 of 1634.7, and has a difference of 365.1. The C16 ion corresponds to S, P and T, which m/z of 87.08, 97.12, and 101.11, respectively, added together gives 285.31. The difference of the value gives 79.79, corresponding to an addition of a phosphate group

    Techniques Used: Mass Spectrometry, Purification, Incubation, In Vitro, SDS Page, Staining, Negative Control, Positive Control, Sequencing, Kinase Assay

    3) Product Images from "Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins"

    Article Title: Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201607095

    Differential effects of Tub on ciliary GPCR trafficking. (A) IMCD3 Flp-In cells stably expressing LAP–TUB isoform b ( Mukhopadhyay et al., 2010 ) were sequentially transfected with 200 nM of the indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted in two experiments, and total counted cells are > 400/condition. Data represent means ± SD. Bars, 5 µm. (B) MBP TUB isoform b and MBP TULP3 immobilized on amylose resin were incubated with PreScission eluates from IFT140 LAP RPE cells ± His TULP3 (1–183 aa; see Materials and methods; Mukhopadhyay et al., 2010 ). LAP, S tag–PreScission-GFP. MBP TUB- and MBP TULP3-bound proteins and corresponding flowthroughs were immunoblotted for S tag (IFT140 S tag ), maltose-binding protein (MBP), and His-tag as indicated. Data are representative of two experiments. (C and D) Embryonic day 16.5, day in vitro (DIV) 8 hippocampal neurons from wild-type (WT) and Tub mice were immunostained for Gpr161/Gpr19 (green), DyLight594-labeled ACIII (red), and Sstr3 (white; C). Gpr161/Gpr19-positive and -negative cilia are marked by arrows (A and C) and arrowheads (C), respectively. Gpr161/Sstr3 coexpressing cells are marked by yellow arrows. The red dot indicates debris under the coverslip. Data represents means ± SD from cultures of two different embryos belonging to each genotype. Total counted cells are > 300 per condition for Gpr161/Sstr3 and > 90 per condition for Gpr19. Bars, 5 µm. Ciliary lengths are 3.2 ± 0.1 µm (wild type) and 3.5 ± 0.1 µm ( Tub ) neurons in experiments with Gpr161 staining and 3.7 ± 0.9 µm (wild type) and 3.5 ± 0.8 µm ( Tub ) in experiments with Gpr19 staining. Data represent means ± SD. n > 50 each. (E) Embryonic day 16.5 DIV5 hippocampal neurons from wild-type mice were transfected with indicated GFP-tagged N terminus (NT) wild-type or non–IFT-A binding ( mut12 ) TULP3 constructs ( Mukhopadhyay et al., 2010 ), fixed at DIV8, and immunostained for Gpr161 and DyLight594-labeled ACIII. GFP-positive cells were quantified for Gpr161-positive cilia from two different coverslips, and total counted neurons are > 24 per condition. (F) Embryonic day 18.5 DIV5 glia from wild-type mice were transfected with constructs as in C, fixed at DIV8, and immunostained for Gpr19 and Arl13b. GFP-positive cells were quantified for Gpr19-positive cilia from cultures from two different mice, and total counted cells are > 110 per condition. (A and D–F) *, P
    Figure Legend Snippet: Differential effects of Tub on ciliary GPCR trafficking. (A) IMCD3 Flp-In cells stably expressing LAP–TUB isoform b ( Mukhopadhyay et al., 2010 ) were sequentially transfected with 200 nM of the indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixation and immunostained for GFP, acetylated tubulin, and DNA. GFP-positive cilia were counted in two experiments, and total counted cells are > 400/condition. Data represent means ± SD. Bars, 5 µm. (B) MBP TUB isoform b and MBP TULP3 immobilized on amylose resin were incubated with PreScission eluates from IFT140 LAP RPE cells ± His TULP3 (1–183 aa; see Materials and methods; Mukhopadhyay et al., 2010 ). LAP, S tag–PreScission-GFP. MBP TUB- and MBP TULP3-bound proteins and corresponding flowthroughs were immunoblotted for S tag (IFT140 S tag ), maltose-binding protein (MBP), and His-tag as indicated. Data are representative of two experiments. (C and D) Embryonic day 16.5, day in vitro (DIV) 8 hippocampal neurons from wild-type (WT) and Tub mice were immunostained for Gpr161/Gpr19 (green), DyLight594-labeled ACIII (red), and Sstr3 (white; C). Gpr161/Gpr19-positive and -negative cilia are marked by arrows (A and C) and arrowheads (C), respectively. Gpr161/Sstr3 coexpressing cells are marked by yellow arrows. The red dot indicates debris under the coverslip. Data represents means ± SD from cultures of two different embryos belonging to each genotype. Total counted cells are > 300 per condition for Gpr161/Sstr3 and > 90 per condition for Gpr19. Bars, 5 µm. Ciliary lengths are 3.2 ± 0.1 µm (wild type) and 3.5 ± 0.1 µm ( Tub ) neurons in experiments with Gpr161 staining and 3.7 ± 0.9 µm (wild type) and 3.5 ± 0.8 µm ( Tub ) in experiments with Gpr19 staining. Data represent means ± SD. n > 50 each. (E) Embryonic day 16.5 DIV5 hippocampal neurons from wild-type mice were transfected with indicated GFP-tagged N terminus (NT) wild-type or non–IFT-A binding ( mut12 ) TULP3 constructs ( Mukhopadhyay et al., 2010 ), fixed at DIV8, and immunostained for Gpr161 and DyLight594-labeled ACIII. GFP-positive cells were quantified for Gpr161-positive cilia from two different coverslips, and total counted neurons are > 24 per condition. (F) Embryonic day 18.5 DIV5 glia from wild-type mice were transfected with constructs as in C, fixed at DIV8, and immunostained for Gpr19 and Arl13b. GFP-positive cells were quantified for Gpr19-positive cilia from cultures from two different mice, and total counted cells are > 110 per condition. (A and D–F) *, P

    Techniques Used: Stable Transfection, Expressing, Transfection, Incubation, Binding Assay, In Vitro, Mouse Assay, Labeling, Staining, Construct

    4) Product Images from "Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas"

    Article Title: Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005384

    IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.
    Figure Legend Snippet: IFT25 interacts physically with IFT27. Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.

    Techniques Used: Purification, In Vitro, Binding Assay, Marker, Labeling, Staining, Western Blot

    5) Product Images from "In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops"

    Article Title: In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops

    Journal: AMB Express

    doi: 10.1186/2191-0855-2-22

    Effect of Chitinolytic enzymes on the structure germ tubes of H. vastatrix after 18 h of treatment application . MBP (Maltose binding protein). Germinating uredospores treated with a. water, showing normal growth; b. BSA (Bovine Serum Albumin) displaying increased cell wall thickness; c. MBP/endochitinase and d. MBP/exochitinase, resulting in germ tubes either broken or shrunken. (Bar = 20 μm).
    Figure Legend Snippet: Effect of Chitinolytic enzymes on the structure germ tubes of H. vastatrix after 18 h of treatment application . MBP (Maltose binding protein). Germinating uredospores treated with a. water, showing normal growth; b. BSA (Bovine Serum Albumin) displaying increased cell wall thickness; c. MBP/endochitinase and d. MBP/exochitinase, resulting in germ tubes either broken or shrunken. (Bar = 20 μm).

    Techniques Used: Binding Assay

    Effects of the MBP/exochitinase, MBP, and BSA at 0.5% (w/w) assayed in artificial coffee-based diets on the mortality of coffee berry borer larvae .
    Figure Legend Snippet: Effects of the MBP/exochitinase, MBP, and BSA at 0.5% (w/w) assayed in artificial coffee-based diets on the mortality of coffee berry borer larvae .

    Techniques Used:

    6) Product Images from "?-Catenin-Vinculin Interaction Functions to Organize the Apical Junctional Complex in Epithelial Cells "

    Article Title: ?-Catenin-Vinculin Interaction Functions to Organize the Apical Junctional Complex in Epithelial Cells

    Journal: The Journal of Cell Biology

    doi:

    In vitro binding of vinculin with αE-catenin. ( A ) Schematic drawing of GST-αE-catenin fusion proteins ( 1–7 ), and of MBP-vinculin fusion proteins (MBP-vinHead and MBP-vinTail). ( B ) Detection of proteins bound to the GST-αE-catenin fusion proteins 2, 3, and 7 ( control ) that had been incubated with chicken gizzard extracts. ( Top ) proteins bound to the GST-fusion proteins were separated by SDS-PAGE, and were visualized by silver staining. Two bands of 130 and 120 kD ( arrowheads ) were precipitated with GST-αE(1–509) ( 2 ), but not with the other constructs. These bands were recognized with anti-vinculin antibodies ( middle ). α-Actinin and spectrin did not bind to any of these constructs, whereas β-catenin was precipitated with 2 as well as with 3 ( bottom ). giz, the original extract of gizzards used for these binding assays; vin, vinculin; αac, α-actinin; spe, spectrin; βca, β-catenin. Positions of molecular markers are 200, 116, and 97 × 10 3 . ( C ) Binding of MBP-vinHead ( vinH ) or MBP-vinTail ( vinT ) to GST-αE-catenin fusion proteins 1–7. Sepharose beads conjugated with these GST-fusion proteins were incubated with a lysate of E . coli expressing the MBP-fusion proteins. Coprecipitated proteins were analyzed by Western blotting with antibodies to vinculin (for MBP-vinHead) or to MBP (for MBP-vinTail; top ). Each sample for electrophoresis contained an equal molar amount of the GST-fusion proteins that had been adjusted before loading on the gel. ( Bottom ) Coomassie blue staining for the GST-αE-catenin fusion proteins 1–7 conjugated to Sepharose beads. Positions of molecular markers are the same as in Fig. 1 A . lys, the original lysate of E . coli used for this binding assay.
    Figure Legend Snippet: In vitro binding of vinculin with αE-catenin. ( A ) Schematic drawing of GST-αE-catenin fusion proteins ( 1–7 ), and of MBP-vinculin fusion proteins (MBP-vinHead and MBP-vinTail). ( B ) Detection of proteins bound to the GST-αE-catenin fusion proteins 2, 3, and 7 ( control ) that had been incubated with chicken gizzard extracts. ( Top ) proteins bound to the GST-fusion proteins were separated by SDS-PAGE, and were visualized by silver staining. Two bands of 130 and 120 kD ( arrowheads ) were precipitated with GST-αE(1–509) ( 2 ), but not with the other constructs. These bands were recognized with anti-vinculin antibodies ( middle ). α-Actinin and spectrin did not bind to any of these constructs, whereas β-catenin was precipitated with 2 as well as with 3 ( bottom ). giz, the original extract of gizzards used for these binding assays; vin, vinculin; αac, α-actinin; spe, spectrin; βca, β-catenin. Positions of molecular markers are 200, 116, and 97 × 10 3 . ( C ) Binding of MBP-vinHead ( vinH ) or MBP-vinTail ( vinT ) to GST-αE-catenin fusion proteins 1–7. Sepharose beads conjugated with these GST-fusion proteins were incubated with a lysate of E . coli expressing the MBP-fusion proteins. Coprecipitated proteins were analyzed by Western blotting with antibodies to vinculin (for MBP-vinHead) or to MBP (for MBP-vinTail; top ). Each sample for electrophoresis contained an equal molar amount of the GST-fusion proteins that had been adjusted before loading on the gel. ( Bottom ) Coomassie blue staining for the GST-αE-catenin fusion proteins 1–7 conjugated to Sepharose beads. Positions of molecular markers are the same as in Fig. 1 A . lys, the original lysate of E . coli used for this binding assay.

    Techniques Used: In Vitro, Binding Assay, Incubation, SDS Page, Silver Staining, Construct, Expressing, Western Blot, Electrophoresis, Staining

    7) Product Images from "Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism"

    Article Title: Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism

    Journal:

    doi: 10.1074/jbc.M109.002519

    Simple Blue TM staining of purified recombinant human MBP-tagged Nmnat 1, mutant, and wild-type Nampts separated by SDS-PAGE. Recombinant human proteins were expressed in E. coli and purified as described under “Experimental Procedures.”
    Figure Legend Snippet: Simple Blue TM staining of purified recombinant human MBP-tagged Nmnat 1, mutant, and wild-type Nampts separated by SDS-PAGE. Recombinant human proteins were expressed in E. coli and purified as described under “Experimental Procedures.”

    Techniques Used: Staining, Purification, Recombinant, Mutagenesis, SDS Page

    8) Product Images from "Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1"

    Article Title: Maintenance of structure and function of mitochondrial Hsp70 chaperones requires the chaperone Hep1

    Journal:

    doi: 10.1038/sj.emboj.7600580

    Hep1 does not increase the ATPase activity and the nucleotide exchange rate of mtHsp70. ( A ) Coomassie-stained gel of purified proteins Hep1-His6 and mtHsp70. ( B ) The ATPase activity of mtHsp70 was determined by the rate of conversion of 32 P-ATP to ADP and 32 P. Samples containing the indicated purified proteins were incubated for the indicated times, subjected to thin layer chromatography and the percentage of hydrolysed ATP was quantified by phosphoimaging. A fusion protein of MBP and Tim14 was used (Tim14). ( C ) Preformed complex of mtHsp70 with 32 P-ATP was incubated at 30°C with excess of unlabelled ATP in the presence of Hep1, Mge1 or both Mge1 and Hep1 as indicated. The fraction of hydrolysed 32 P-ATP was determined at the indicated time points as in (B).
    Figure Legend Snippet: Hep1 does not increase the ATPase activity and the nucleotide exchange rate of mtHsp70. ( A ) Coomassie-stained gel of purified proteins Hep1-His6 and mtHsp70. ( B ) The ATPase activity of mtHsp70 was determined by the rate of conversion of 32 P-ATP to ADP and 32 P. Samples containing the indicated purified proteins were incubated for the indicated times, subjected to thin layer chromatography and the percentage of hydrolysed ATP was quantified by phosphoimaging. A fusion protein of MBP and Tim14 was used (Tim14). ( C ) Preformed complex of mtHsp70 with 32 P-ATP was incubated at 30°C with excess of unlabelled ATP in the presence of Hep1, Mge1 or both Mge1 and Hep1 as indicated. The fraction of hydrolysed 32 P-ATP was determined at the indicated time points as in (B).

    Techniques Used: Activity Assay, Staining, Purification, Incubation, Thin Layer Chromatography

    9) Product Images from "Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17"

    Article Title: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17

    Journal: Nature Communications

    doi: 10.1038/ncomms15406

    Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .
    Figure Legend Snippet: Lpg1137 is a serine protease localized in MAM/mitochondria. ( a ) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. ( b ) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. ( c , d ) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the ( c ) indicated antibodies. Alternatively, cells were fixed and stained with ( d ) an anti-Stx17 antibody. Scale bar, 5 μm. ( e ) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7 .

    Techniques Used: Transfection, Plasmid Preparation, Fractionation, Mass Spectrometry, Incubation, Staining

    10) Product Images from "Overexpression of the PeaT1 Elicitor Gene from Alternaria tenuissima Improves Drought Tolerance in Rice Plants via Interaction with a Myo-Inositol Oxygenase"

    Article Title: Overexpression of the PeaT1 Elicitor Gene from Alternaria tenuissima Improves Drought Tolerance in Rice Plants via Interaction with a Myo-Inositol Oxygenase

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.00970

    PeaT1 interacts with OsMIOX. (A) Results of a yeast two-hybrid assay involving BD-PeaT1 and AD-OsMIOX. The transformed yeast cells were plated on SD/-Leu/-Trp medium (left) and SD/-Leu/-Trp/-His medium supplemented with 50 mM 3-AT (middle). The results of the X-gal assay are also provided (right). (B) Purified proteins stained with Coomassie Brilliant Blue. (C) A solution consisting of PeaT1-GST bound to the GST-binding resin was incubated with an equal volume of cell lysate containing OsMIOX-MBP or MBP. The binding of OsMIOX-MBP or MBP to the GST-binding resin before (Input) and after (Pull-down) washes was examined in a western blot involving the anti-MBP antibody. The same protein gel was used for a western blot involving the anti-GST antibody to verify that equal amounts of GST-PeaT1 were present in all protein mixtures. ∗ Represents the target protein band.
    Figure Legend Snippet: PeaT1 interacts with OsMIOX. (A) Results of a yeast two-hybrid assay involving BD-PeaT1 and AD-OsMIOX. The transformed yeast cells were plated on SD/-Leu/-Trp medium (left) and SD/-Leu/-Trp/-His medium supplemented with 50 mM 3-AT (middle). The results of the X-gal assay are also provided (right). (B) Purified proteins stained with Coomassie Brilliant Blue. (C) A solution consisting of PeaT1-GST bound to the GST-binding resin was incubated with an equal volume of cell lysate containing OsMIOX-MBP or MBP. The binding of OsMIOX-MBP or MBP to the GST-binding resin before (Input) and after (Pull-down) washes was examined in a western blot involving the anti-MBP antibody. The same protein gel was used for a western blot involving the anti-GST antibody to verify that equal amounts of GST-PeaT1 were present in all protein mixtures. ∗ Represents the target protein band.

    Techniques Used: Y2H Assay, Transformation Assay, Purification, Staining, Binding Assay, Incubation, Western Blot

    11) Product Images from "Kebab: Kinetochore and EB1 Associated Basic Protein That Dynamically Changes Its Localisation during Drosophila Mitosis"

    Article Title: Kebab: Kinetochore and EB1 Associated Basic Protein That Dynamically Changes Its Localisation during Drosophila Mitosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024174

    Kebab has EB1 binding motifs, an atypical CH domain and a coiled-coil region. (A) A diagram of the Kebab protein structure. (B) Similarity between the CH domains of human Ndc80 (hNdc80), D. melanogaster Ndc80 (dNdc80) and D. melanogaster Kebab (dKebab). The identical residues between two proteins were shown in bold blue letters. The asterisks indicate the residues important for the microtubule binding in human Ndc80. (C) A series of truncations and mutations tested for their localisation. KC and MT indicates the degree of localisation to kinetochores and microtubules. (++) full localisation, (+) weak localisation, (±) a trace of localisation, (−) no localisation. (D) EB1 binding assay of Kebab with mutated SxIP motifs. Radiolabelled proteins were in vitro translated and mixed with beads coupled with MBP and MBP-EB1. Pull-down fractions were run along with the original input (25% of pull-down fractions) and radiolabelled Kebab was detected by autoradiograph. Specific EB1 binding activity by this assay is indicated together with kinetochore or microtubule localisation (KC or MT). (E) Kinetochore localisation of a full-length GFP-Kebab and Kebab with both EB1 binding motifs mutated (ΔIPs). (F) Kinetochore localisation in EB1 depleted cells. Bar = 10 µm.
    Figure Legend Snippet: Kebab has EB1 binding motifs, an atypical CH domain and a coiled-coil region. (A) A diagram of the Kebab protein structure. (B) Similarity between the CH domains of human Ndc80 (hNdc80), D. melanogaster Ndc80 (dNdc80) and D. melanogaster Kebab (dKebab). The identical residues between two proteins were shown in bold blue letters. The asterisks indicate the residues important for the microtubule binding in human Ndc80. (C) A series of truncations and mutations tested for their localisation. KC and MT indicates the degree of localisation to kinetochores and microtubules. (++) full localisation, (+) weak localisation, (±) a trace of localisation, (−) no localisation. (D) EB1 binding assay of Kebab with mutated SxIP motifs. Radiolabelled proteins were in vitro translated and mixed with beads coupled with MBP and MBP-EB1. Pull-down fractions were run along with the original input (25% of pull-down fractions) and radiolabelled Kebab was detected by autoradiograph. Specific EB1 binding activity by this assay is indicated together with kinetochore or microtubule localisation (KC or MT). (E) Kinetochore localisation of a full-length GFP-Kebab and Kebab with both EB1 binding motifs mutated (ΔIPs). (F) Kinetochore localisation in EB1 depleted cells. Bar = 10 µm.

    Techniques Used: Binding Assay, In Vitro, Autoradiography, Activity Assay

    Kebab interacts with EB1. Cell extract from a stable cell line expressing Kebab-GFP was incubated with bacterially produced MBP and MBP-EB1. MBP and MBP-EB1 were pulled down and subjected to western blot using an anti-Kebab antibody (the upper panel). One twentieth of the cell extract was run, relative to the pull-down fractions. Protein staining of the same membrane is shown in the lower panel. Kebab-GFP is specifically pulled down with EB1.
    Figure Legend Snippet: Kebab interacts with EB1. Cell extract from a stable cell line expressing Kebab-GFP was incubated with bacterially produced MBP and MBP-EB1. MBP and MBP-EB1 were pulled down and subjected to western blot using an anti-Kebab antibody (the upper panel). One twentieth of the cell extract was run, relative to the pull-down fractions. Protein staining of the same membrane is shown in the lower panel. Kebab-GFP is specifically pulled down with EB1.

    Techniques Used: Stable Transfection, Expressing, Incubation, Produced, Western Blot, Staining

    12) Product Images from "The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana"

    Article Title: The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071078

    Analysis of the AtRDUF1 protein. (A) Alignment of the RING finger domains of the AtRDUF1 homologs in Arabidopsis . Black and gray indicate 100% and ≥50% identities, respectively. (B) Subcellular localization of AtRDUF1:GFP fusion protein in Arabidopsis leaf protoplast cells. Bars represent 20 μm. The green and blue fluorescenece are GFP and 4′,6-diamidino-2-phenylindole (DAPI) signals, respectively. (C) Verification of E3 ligase activity of AtRDUF1 by in vitro autoubiquitination assay. CH/Y represents the mutant form of the MBP:AtRDUF1 fusion protein, with substitution of metal ligand positions Cys-3, His-4, and His-5 of the RING motif with Tyr. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Nichel-HRP (Ub), the nickel-horseradish peroxidase used to detect His-tagged ubiquitin. Anti-MBP, the anti-MBP antibody to detect maltose fusion proteins.
    Figure Legend Snippet: Analysis of the AtRDUF1 protein. (A) Alignment of the RING finger domains of the AtRDUF1 homologs in Arabidopsis . Black and gray indicate 100% and ≥50% identities, respectively. (B) Subcellular localization of AtRDUF1:GFP fusion protein in Arabidopsis leaf protoplast cells. Bars represent 20 μm. The green and blue fluorescenece are GFP and 4′,6-diamidino-2-phenylindole (DAPI) signals, respectively. (C) Verification of E3 ligase activity of AtRDUF1 by in vitro autoubiquitination assay. CH/Y represents the mutant form of the MBP:AtRDUF1 fusion protein, with substitution of metal ligand positions Cys-3, His-4, and His-5 of the RING motif with Tyr. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Nichel-HRP (Ub), the nickel-horseradish peroxidase used to detect His-tagged ubiquitin. Anti-MBP, the anti-MBP antibody to detect maltose fusion proteins.

    Techniques Used: Activity Assay, In Vitro, Mutagenesis, Marker

    13) Product Images from "Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations"

    Article Title: Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004928

    Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction. (A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.
    Figure Legend Snippet: Point mutations of FLNa and FilGAP confirms the in silico model of their binding interaction. (A) A point mutation in FLNa (M2474E) abolishes the complexing of FLNa and FilGAP. The upper panel shows amylose beads coated with MBP-FilGAP649-748 pulls down wild-type FLNa but not FLNaM2474E. The lower panel shows wild-type (WT) FLAG-FLNa immobilized on FLAG-specific mAb immobilized on agarose beads, but not FLAG-FLNaM2474E, pull down full-length FilGAP. (B) FLAG-FLNa does not pull point mutants of FilGAP at G730W and V734Y. T728V mutation has no effect on the interaction.

    Techniques Used: In Silico, Binding Assay, Mutagenesis

    Localization of FLNa-FilGAP binding site. (A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.
    Figure Legend Snippet: Localization of FLNa-FilGAP binding site. (A) Schematic representation of FilGAP and its truncation series. The pleckstrin-homology (PH), GTPase-activating protein (GAP), and coiled-coil (CC) domains predicted by EMBnet COILS are shown. Right panel shows binding of FLAG-FLNa to GST-FilGAP fragments illustrated in the left panel. Their interactions were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to GST. (B) FilGAP fragments were fused to MBP-His-tag and their binding to FLAG-FLNa were analyzed by pull-down using FLAG-specific mAb immobilized on beads. Bound protein was detected by immunoblotting using rabbit pAb to MBP (C) His-tag FilGAP, or FilGAP lacking residues 649–725 (50 nM), were mixed with increasing amounts of FLAG-FLNa and immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using anti-His-tag mouse mAb conjugated with horse radish peroxidase (upper panel). The lower panel shows proteins visualized by CBB staining. (D) Molecular weight calibration curve obtained with a Superose 6 10/300 gel filtration column. Molecular size standards (open circle) used were thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa). Colored circles indicate the sizes of His-FilGAP, His-FilGAP lacking residues 649–725 or FilGAP truncates fused to MBP-His-tag.

    Techniques Used: Binding Assay, Immunoprecipitation, Staining, Molecular Weight, Filtration

    FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP. (A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.
    Figure Legend Snippet: FLNa dimerization and hinge-2 are essential for high avidity binding to FilGAP. (A) Full-length FilGAP was pulled down with increasing amounts of wild-type and deletion mutants (Ä23; deletion of IgFLNa23, ÄH2; deletion of FLNa hinge-2, Ä24; deletion of IgFLNa24) of FLNa tagged to FLAG immunoprecipitated with FLAG-specific mAb immobilized on agarose. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) Left panel shows purified MBP-FilGAP649-748, IgFLNa23-24, and IgFLNa23-24 ÄH2 separated on SDS-PAGE and stained with CBB. Right panel; IgFLNa23-24 or IgFLNa23-24 ÄH2 were pulled down with amylose beads coated with increasing amounts of the MBP-FilGAP649-748. Proteins were visualized by CBB staining.

    Techniques Used: Binding Assay, Immunoprecipitation, Purification, SDS Page, Staining

    FilGAP specifically interacts with FLNa isoform. (A) Full-length FLNa, but not FLNb, pulls down FilGAP. Increasing amounts of either FLNa or FLNb were incubated with FilGAP and immunoprecipitated with mAbs to FLNa or FLNb. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) MBP-FilGAP649-748 specifically binds the C-terminal of FLNa, but not FLNb or FLNc. Equal amounts of repeats 23–24 of FLNa, b, or c (0.2 ìM) were pulled down with increasing amounts of MBP-FilGAP659-748. Proteins were visualized by CBB staining (top and bottom). The top CBB-stained gel was destained and restained with silver (middle). (C) Sequence alignment of the C–E strands of the IgFLN23 isoforms. FLNa A2461T, M2474E and Y2483H point mutants do not interact with FilGAP as shown in Figures 4A , 5A and 6E . (D) Model of the IgFLNa23-FilGAP complex. Residues mutated in this study and some critical residues for their interaction are indicated. The purple dotted line shows the possible stabilizing hydrogen-bond between Tyr2483 and Thr733. (E) A point mutation of FLNa at Ala2461 to Thr or Tyr2483 to His are sufficient to abolish the complexing of FLNa and FilGAP. Full-length FilGAP (input: 10 nM constant) was pulled down with increasing amount of GST-IgFLNa20-24 immobilized on glutathione beads in a dose-dependent fashion. Mutations corresponding to A2461T or Y2483H in FLNa, but not D2467E, disrupt FilGAP binding. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. GST-FLNa constructs were detected by CBB staining.
    Figure Legend Snippet: FilGAP specifically interacts with FLNa isoform. (A) Full-length FLNa, but not FLNb, pulls down FilGAP. Increasing amounts of either FLNa or FLNb were incubated with FilGAP and immunoprecipitated with mAbs to FLNa or FLNb. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. (B) MBP-FilGAP649-748 specifically binds the C-terminal of FLNa, but not FLNb or FLNc. Equal amounts of repeats 23–24 of FLNa, b, or c (0.2 ìM) were pulled down with increasing amounts of MBP-FilGAP659-748. Proteins were visualized by CBB staining (top and bottom). The top CBB-stained gel was destained and restained with silver (middle). (C) Sequence alignment of the C–E strands of the IgFLN23 isoforms. FLNa A2461T, M2474E and Y2483H point mutants do not interact with FilGAP as shown in Figures 4A , 5A and 6E . (D) Model of the IgFLNa23-FilGAP complex. Residues mutated in this study and some critical residues for their interaction are indicated. The purple dotted line shows the possible stabilizing hydrogen-bond between Tyr2483 and Thr733. (E) A point mutation of FLNa at Ala2461 to Thr or Tyr2483 to His are sufficient to abolish the complexing of FLNa and FilGAP. Full-length FilGAP (input: 10 nM constant) was pulled down with increasing amount of GST-IgFLNa20-24 immobilized on glutathione beads in a dose-dependent fashion. Mutations corresponding to A2461T or Y2483H in FLNa, but not D2467E, disrupt FilGAP binding. Bound FilGAP was detected by immunoblotting using rabbit pAbs to FilGAP. GST-FLNa constructs were detected by CBB staining.

    Techniques Used: Incubation, Immunoprecipitation, Staining, Sequencing, Mutagenesis, Binding Assay, Construct

    14) Product Images from "Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos"

    Article Title: Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201408064

    Phospho–SPAT-1 and phospho-Bora promote PLK-1/Plk1 phosphorylation on its activator T loop by the Aurora A kinase. (A) Flow chart of the assay used to test the role of Bora/SPAT-1 phosphorylation by CyclinB/Cdk1 in Plk1 phosphorylation by Aurora A. (B) The T loop of Plk1 is evolutionarily conserved. Sequence alignment of the T-loop region of Plk1 from various species, using PRALINE ( H. s , H. sapiens ; M. m , Mus musculus ; X. l , X. laevis ; D. r , Danio rerio ; C. e , C. elegans ; D. m , D. melanogaster ). Invariant residues are highlighted in red. An arrow marks the activating T210 phosphorylation of human Plk1, which corresponds to T194 in C.e. PLK-1. (C) Western blot of WT and T194A 6×(His)–PLK-1 mutant purified from insect Sf9 cells with the anti-Plk1 pT210 (top) and PLK-1 antibody (bottom). (D) The phospho-specific T210 Plk1 antibody specifically recognizes pT194 in C.e. PLK-1. 6×(His)–PLK-1 WT purified from insect Sf9 cells was incubated with λ phosphatase (λ PPase, +) or heat-inactivated phosphatase (#) and analyzed by SDS-PAGE and Western blotting using the anti-Plk1 phospho-T210 (top) and PLK-1 antibodies (bottom). (E) Western blot showing the result of the kinase assay of 6×(His)-Plk1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP-Bora (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and Plk1 (middle) antibodies. (F) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP–SPAT-1 (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies. (G) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lane 1), MBP–SPAT-1 WT (lane 2), or MBP–SPAT-1 13A mutant phosphorylated by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies.
    Figure Legend Snippet: Phospho–SPAT-1 and phospho-Bora promote PLK-1/Plk1 phosphorylation on its activator T loop by the Aurora A kinase. (A) Flow chart of the assay used to test the role of Bora/SPAT-1 phosphorylation by CyclinB/Cdk1 in Plk1 phosphorylation by Aurora A. (B) The T loop of Plk1 is evolutionarily conserved. Sequence alignment of the T-loop region of Plk1 from various species, using PRALINE ( H. s , H. sapiens ; M. m , Mus musculus ; X. l , X. laevis ; D. r , Danio rerio ; C. e , C. elegans ; D. m , D. melanogaster ). Invariant residues are highlighted in red. An arrow marks the activating T210 phosphorylation of human Plk1, which corresponds to T194 in C.e. PLK-1. (C) Western blot of WT and T194A 6×(His)–PLK-1 mutant purified from insect Sf9 cells with the anti-Plk1 pT210 (top) and PLK-1 antibody (bottom). (D) The phospho-specific T210 Plk1 antibody specifically recognizes pT194 in C.e. PLK-1. 6×(His)–PLK-1 WT purified from insect Sf9 cells was incubated with λ phosphatase (λ PPase, +) or heat-inactivated phosphatase (#) and analyzed by SDS-PAGE and Western blotting using the anti-Plk1 phospho-T210 (top) and PLK-1 antibodies (bottom). (E) Western blot showing the result of the kinase assay of 6×(His)-Plk1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP-Bora (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and Plk1 (middle) antibodies. (F) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lanes 1 and 2) or MBP–SPAT-1 (lanes 3 and 4) phosphorylated (+) or not phosphorylated (−) by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies. (G) Western blot showing the result of the kinase assay of 6×(His)–PLK-1 by 6×(His)–Aurora A incubated with MBP (lane 1), MBP–SPAT-1 WT (lane 2), or MBP–SPAT-1 13A mutant phosphorylated by CyclinB/Cdk1. Blots were probed with MBP (top), anti-pT210 (bottom), and PLK-1 (middle) antibodies.

    Techniques Used: Flow Cytometry, Sequencing, Western Blot, Mutagenesis, Purification, Incubation, SDS Page, Kinase Assay

    SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[ 32 P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32 P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.
    Figure Legend Snippet: SPAT-1 phosphorylation by Cdk1 promotes the interaction between SPAT-1 and PLK-1. (A, top) Embryonic extracts of the indicated genotypes analyzed by Western blotting using SPAT-1 antibodies. (bottom) Tubulin is used as a loading control. 25 µg (lanes 1, 3, 5, and 7) and 50 µg (lanes 2, 4, 6, and 8) of each protein extract were loaded to visualize the modified forms. (B) MBP–SPAT-1 or MBP incubated with CyclinB/Cdk1 kinase in the presence of γ-[ 32 P]ATP. (right) Autoradiograph of the SDS-PAGE gel showing 32 P incorporation in MBP–SPAT-1 but not MBP. (left) Coomassie staining of the same SDS-PAGE gel. (C) Western blot analysis of PLK-1 immunoprecipitates (IP PLK-1) from control (lane 3) or cdk-1(RNAi) (lane 4) embryonic extracts analyzed with SPAT-1 (top) and PLK-1 antibodies (middle). (bottom) Actin was used as a loading control. 10 µg (1:40) of the total extracts (Ext.; lanes 1 and 2) and the flow through (FT) of the immunoprecipitates (lanes 5 and 6) were loaded for comparison. The asterisk marks the phosphorylated SPAT-1 forms that are present in the PLK-1 immunoprecipitation. (D) In vitro assay used to test Cdk1 dependency of the interaction between SPAT-1 and PLK-1. On the left, we show a flow chart describing the assay; on the right, we show the Western blot analysis. Strep–SPAT-1 protein produced in insect Sf9 cells was immobilized on Strep-Tactin Sepharose beads, dephosphorylated with λ phosphatase (λ PPase), and incubated with CylinB/Cdk1 in the presence (+) or absence (−) of ATP. After washing the kinase and ATP, full-length 6×(His)–PLK-1 was added (+) for a typical pull-down experiment. (right) Strep–SPAT-1 was eluted with desthiobiotin, and the elutions were analyzed by SDS-PAGE and Western blotting using PLK-1 and SPAT-1 antibodies.

    Techniques Used: Western Blot, Modification, Incubation, Autoradiography, SDS Page, Staining, Flow Cytometry, Immunoprecipitation, In Vitro, Produced

    15) Product Images from "MHF1-2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination"

    Article Title: MHF1-2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination

    Journal: Open Biology

    doi: 10.1098/rsob.130102

    MHF interacts with Fml1. ( a ) Schematic of Fml1 and the various truncated forms of it used in ( b ) and ( c ). The position of the MBP fusion, DNA helicase motifs (blue bars), and region encompassing the site of MHF interaction (red box) are shown. The numbers refer to amino acid positions. ( b–d ) Western blots showing the amount of His-tagged Mhf2 retained on amylose resin that has been pre-incubated with the indicated MBP-Fml1 fragment (the numbers refer to amino acid positions). ( e ) Amino acids 670–690 in Fml1 with the mutations tested in ( f ) indicated. ( f ) Western blot showing the retention of His-tagged Mhf2 on amylose resin pre-incubated with MBP-Fml1 650–690 or its mutant derivatives 1–3. ( g ) Western blot showing that full-length Fml1 AAA fused to MBP fails to retain HisMhf2 on amylose resin. ( h ) SDS-PAGE analysis of gel filtration fractions 32–38 from the purification of the Fml1 576–725 –MHF complex. The gel was stained with Coomassie blue. ( i ) EMSA comparing the ability of MHF and the Fml1 576–725 –MHF complex to bind linear dsDNA. The amounts of protein are 49 nM (lanes b and f), 98 nM (lanes c and g), 490 nM (lanes d and h) and 980 nM (lanes e and i).
    Figure Legend Snippet: MHF interacts with Fml1. ( a ) Schematic of Fml1 and the various truncated forms of it used in ( b ) and ( c ). The position of the MBP fusion, DNA helicase motifs (blue bars), and region encompassing the site of MHF interaction (red box) are shown. The numbers refer to amino acid positions. ( b–d ) Western blots showing the amount of His-tagged Mhf2 retained on amylose resin that has been pre-incubated with the indicated MBP-Fml1 fragment (the numbers refer to amino acid positions). ( e ) Amino acids 670–690 in Fml1 with the mutations tested in ( f ) indicated. ( f ) Western blot showing the retention of His-tagged Mhf2 on amylose resin pre-incubated with MBP-Fml1 650–690 or its mutant derivatives 1–3. ( g ) Western blot showing that full-length Fml1 AAA fused to MBP fails to retain HisMhf2 on amylose resin. ( h ) SDS-PAGE analysis of gel filtration fractions 32–38 from the purification of the Fml1 576–725 –MHF complex. The gel was stained with Coomassie blue. ( i ) EMSA comparing the ability of MHF and the Fml1 576–725 –MHF complex to bind linear dsDNA. The amounts of protein are 49 nM (lanes b and f), 98 nM (lanes c and g), 490 nM (lanes d and h) and 980 nM (lanes e and i).

    Techniques Used: Western Blot, Incubation, Mutagenesis, SDS Page, Filtration, Purification, Staining

    16) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M113.527937

    RING finger of Pex12p enhances ubiquitin ligase activity of Pex10pC. A , in vitro ubiquitination assay was performed with MBP-Pex10pC, MBP-Pex12pC, MBP-Pex2pC-HA2 , and MBP-HA2 as indicated at the top in the presence of E2, UbcH5C. The reaction mixtures were analyzed by immunoblotting with antibodies to Pex10p, Pex12p, and HA. Solid , open , and shaded arrowheads , respective authentic MBP-RING peroxins, MBP-HA2 , and the self-ubiquitinated form of MBP-Pex2pC-HA2 , respectively. B , ubiquitination assays were likewise performed as in A with MBP-Pex10pC, MBP-Pex12pC, and MBP-Pex12pC-C304S (a RING mutant of Pex12pC), as indicated.
    Figure Legend Snippet: RING finger of Pex12p enhances ubiquitin ligase activity of Pex10pC. A , in vitro ubiquitination assay was performed with MBP-Pex10pC, MBP-Pex12pC, MBP-Pex2pC-HA2 , and MBP-HA2 as indicated at the top in the presence of E2, UbcH5C. The reaction mixtures were analyzed by immunoblotting with antibodies to Pex10p, Pex12p, and HA. Solid , open , and shaded arrowheads , respective authentic MBP-RING peroxins, MBP-HA2 , and the self-ubiquitinated form of MBP-Pex2pC-HA2 , respectively. B , ubiquitination assays were likewise performed as in A with MBP-Pex10pC, MBP-Pex12pC, and MBP-Pex12pC-C304S (a RING mutant of Pex12pC), as indicated.

    Techniques Used: Activity Assay, In Vitro, Ubiquitin Assay, Hemagglutination Assay, Mutagenesis

    Pex5p is a potential substrate of Pex10p·Pex12p ubiquitin ligase complex. A , in vitro ubiquitination of Pex5p. Wild-type MBP-Pex10pC, wild type ( W ) or C304S mutant ( M ) of MBP-Pex12pC, and Pex5pL were assessed by an in vitro ubiquitination assay as in C. B , K1/FLP10-derived FLAG-Pex10p ubiquitinates Pex5p and Pex12p. The in vitro ubiquitination assay was performed as in A , using wild type ( W ) or C304S mutant ( M ) of MBP-Pex12pC, Pex5pL, and an E3, FLAG-Pex10p immunoprecipitated from K1/FLP10 cells. Ubiquitinated Pex5p bands are designated with a bracket. C , in vitro ubiquitination assay was performed with MBP-fused full-length Pex10p (MBP-10pFull) and Pex12p (MBP-12pFull), in the absence (−) or presence (+) of Pex5pL. Arrowhead and bracket , unmodified and ubiquitinated Pex5pL, respectively. D , in vitro ubiquitination assay was performed as in lane 9 in C , except for using wild-type ubiquitin ( WT ) or its mutants, K48R, K63R, and K0. Arrowheads , unmodified Pex5pL ( top ), MBP-Pex10pFull ( middle ), and MBP-Pex12pFull ( bottom ). Note that apparently monoubiquitinated Pex5p and Pex10p were discernible in all types of K-mutants of ubiquitin. E , an in vitro ubiquitination assay was performed as in A using MBP-Pex10pC, MBP-Pex12pC, MBP-Pex2pC, and Pex5pL variants, as indicated, where DTT was omitted from the reaction mixture. The reaction was terminated by incubation in Laemmli SDS-PAGE sample buffer in the absence of DTT at 50 °C (−) for 10 min ( lanes 1–5 and 11–15 ) or presence of 0.1 m DTT at 100 °C (+) for 5 min ( lanes 6–10 and 16–20 ). Note that DTT-sensitive ubiquitin modification of Pex5pL was hardly detectable with any single RING peroxin or any combination of them. WB , Western blot.
    Figure Legend Snippet: Pex5p is a potential substrate of Pex10p·Pex12p ubiquitin ligase complex. A , in vitro ubiquitination of Pex5p. Wild-type MBP-Pex10pC, wild type ( W ) or C304S mutant ( M ) of MBP-Pex12pC, and Pex5pL were assessed by an in vitro ubiquitination assay as in C. B , K1/FLP10-derived FLAG-Pex10p ubiquitinates Pex5p and Pex12p. The in vitro ubiquitination assay was performed as in A , using wild type ( W ) or C304S mutant ( M ) of MBP-Pex12pC, Pex5pL, and an E3, FLAG-Pex10p immunoprecipitated from K1/FLP10 cells. Ubiquitinated Pex5p bands are designated with a bracket. C , in vitro ubiquitination assay was performed with MBP-fused full-length Pex10p (MBP-10pFull) and Pex12p (MBP-12pFull), in the absence (−) or presence (+) of Pex5pL. Arrowhead and bracket , unmodified and ubiquitinated Pex5pL, respectively. D , in vitro ubiquitination assay was performed as in lane 9 in C , except for using wild-type ubiquitin ( WT ) or its mutants, K48R, K63R, and K0. Arrowheads , unmodified Pex5pL ( top ), MBP-Pex10pFull ( middle ), and MBP-Pex12pFull ( bottom ). Note that apparently monoubiquitinated Pex5p and Pex10p were discernible in all types of K-mutants of ubiquitin. E , an in vitro ubiquitination assay was performed as in A using MBP-Pex10pC, MBP-Pex12pC, MBP-Pex2pC, and Pex5pL variants, as indicated, where DTT was omitted from the reaction mixture. The reaction was terminated by incubation in Laemmli SDS-PAGE sample buffer in the absence of DTT at 50 °C (−) for 10 min ( lanes 1–5 and 11–15 ) or presence of 0.1 m DTT at 100 °C (+) for 5 min ( lanes 6–10 and 16–20 ). Note that DTT-sensitive ubiquitin modification of Pex5pL was hardly detectable with any single RING peroxin or any combination of them. WB , Western blot.

    Techniques Used: In Vitro, Mutagenesis, Ubiquitin Assay, Derivative Assay, Immunoprecipitation, Incubation, SDS Page, Modification, Western Blot

    RING finger of Pex10p shows self-ubiquitinating activity in vitro and is required for its complementing activity of pex10 fibroblasts in vivo . A , schematic diagram of domain structure of RING peroxins. Gray boxes , transmembrane domains; black boxes labeled R , RING finger motifs. B , modification of MBP-Pex10pC in vitro . An in vitro ubiquitination assay was performed using nine E2 enzymes, each with MBP-Pex10pC ( top ), MBP-Pex12pC ( middle ), and MBP-Pex2pC ( bottom ) as described under “Materials and Methods.” Reaction mixtures were assessed by immunoblotting with anti-MBP antibody. C , self-ubiquitination of MBP-Pex10pC depends on its RING finger. Top , alignment of amino acid sequences of the RING finger in Pex10p from HsPEX10 ( Homo sapiens ), HpPEX10 ( Hansenula polymorpha ), and PpPEX10 ( P. pastoris ). The eight amino acid residues, C3 HC4 , conserved in the RING finger are shaded ; mutations used in this study are indicated below. Bottom , an in vitro ubiquitination assay was performed with wild-type and mutant MBP-Pex10pC in the presence of E2 UbcH5C. The reaction mixtures were verified by immunoblotting with antibodies to Pex10p ( left ) and Ub ( right ). Solid arrowhead , non-modified MBP-Pex10pC. D , complementing activity of Pex10p RING mutants. Fibroblasts from a PEX10 -defective PBD patient (PBDB-01) were transfected with plasmids encoding FLAG-Pex10p variants. At 48 h after the transfection, cells were immunostained with anti-PTS1 antibody. Bar , 10 μm. E , localization of Pex10p RING mutants. CHO-K1 cells were transfected with plasmids encoding wild-type FLAG-Pex10p and the RING mutant C273A. At 24 h post-transfection, cells were immunostained with antibodies to FLAG ( a and c ) and PTS1 ( b and d ). Bar , 10 μm. F , stability of Pex10p RING mutants. CHO-K1 cells were transfected as in E and then lysed and analyzed by immunoblotting ( WB ) with the antibodies indicated at the bottom. Arrowheads , exogenously expressed FLAG-Pex10p variants.
    Figure Legend Snippet: RING finger of Pex10p shows self-ubiquitinating activity in vitro and is required for its complementing activity of pex10 fibroblasts in vivo . A , schematic diagram of domain structure of RING peroxins. Gray boxes , transmembrane domains; black boxes labeled R , RING finger motifs. B , modification of MBP-Pex10pC in vitro . An in vitro ubiquitination assay was performed using nine E2 enzymes, each with MBP-Pex10pC ( top ), MBP-Pex12pC ( middle ), and MBP-Pex2pC ( bottom ) as described under “Materials and Methods.” Reaction mixtures were assessed by immunoblotting with anti-MBP antibody. C , self-ubiquitination of MBP-Pex10pC depends on its RING finger. Top , alignment of amino acid sequences of the RING finger in Pex10p from HsPEX10 ( Homo sapiens ), HpPEX10 ( Hansenula polymorpha ), and PpPEX10 ( P. pastoris ). The eight amino acid residues, C3 HC4 , conserved in the RING finger are shaded ; mutations used in this study are indicated below. Bottom , an in vitro ubiquitination assay was performed with wild-type and mutant MBP-Pex10pC in the presence of E2 UbcH5C. The reaction mixtures were verified by immunoblotting with antibodies to Pex10p ( left ) and Ub ( right ). Solid arrowhead , non-modified MBP-Pex10pC. D , complementing activity of Pex10p RING mutants. Fibroblasts from a PEX10 -defective PBD patient (PBDB-01) were transfected with plasmids encoding FLAG-Pex10p variants. At 48 h after the transfection, cells were immunostained with anti-PTS1 antibody. Bar , 10 μm. E , localization of Pex10p RING mutants. CHO-K1 cells were transfected with plasmids encoding wild-type FLAG-Pex10p and the RING mutant C273A. At 24 h post-transfection, cells were immunostained with antibodies to FLAG ( a and c ) and PTS1 ( b and d ). Bar , 10 μm. F , stability of Pex10p RING mutants. CHO-K1 cells were transfected as in E and then lysed and analyzed by immunoblotting ( WB ) with the antibodies indicated at the bottom. Arrowheads , exogenously expressed FLAG-Pex10p variants.

    Techniques Used: Activity Assay, In Vitro, In Vivo, Labeling, Modification, Ubiquitin Assay, Mutagenesis, Transfection, Western Blot

    17) Product Images from "Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles"

    Article Title: Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles

    Journal:

    doi: 10.1105/tpc.113.113704

    Interactions between Myosin XI-K and Proteins Encoded by Arabidopsis Genes AT1go88oo ( MyoB1 ), At1g70750 ( MyoB2 ), and At5g16720 ( MyoB3 ). (A) assay on SD/-Leu/-Trp/-His/-Ade plates supplemented with X-α-Gal. The combinations of bait and prey proteins used in each sector of the Petri dish were as follows: 1, XI-K-GTD + MyoB1; 2, XI-K-GTD + MyoB2; 3, XI-K-GTD + MyoB3; 4, MyoB3 + GFP; 5, MyoB2 + GFP; 6, MyoB1 + GFP. (B) Pull-down assay showing specific binding of MyoB1-YFP and MyoB2-YFP to immobilized XI-K (MBP-GTD) but not to immobilized GUS (MBP-GUS). GFP-GUS provides specificity control via binding to MBP-GUS (GUS forms dimers) but not to MBP-GTD. (C) Coimmunoprecipitation of the MyoB1-YFP–myosin XI-K complexes formed in vivo. α-GFP:AG, GFP-specific monoclonal antibody immobilized on agarose beads; AG, uncharged agarose beads control. (D) Pull-down assay showing specific binding of the MyoB2 DUF593 domain (dufMyoB2-GFP) to immobilized XI-K (MBP-GTD). Free GFP and N-terminal fragment of MyoB2 (NMyoB2-GFP) provide binding specificity controls. Arrows at the left show protein marker positions with their molecular mass in kilodaltons. Red asterisks mark full-size protein bands; bands of lower molecular mass probably correspond to protein degradation products.
    Figure Legend Snippet: Interactions between Myosin XI-K and Proteins Encoded by Arabidopsis Genes AT1go88oo ( MyoB1 ), At1g70750 ( MyoB2 ), and At5g16720 ( MyoB3 ). (A) assay on SD/-Leu/-Trp/-His/-Ade plates supplemented with X-α-Gal. The combinations of bait and prey proteins used in each sector of the Petri dish were as follows: 1, XI-K-GTD + MyoB1; 2, XI-K-GTD + MyoB2; 3, XI-K-GTD + MyoB3; 4, MyoB3 + GFP; 5, MyoB2 + GFP; 6, MyoB1 + GFP. (B) Pull-down assay showing specific binding of MyoB1-YFP and MyoB2-YFP to immobilized XI-K (MBP-GTD) but not to immobilized GUS (MBP-GUS). GFP-GUS provides specificity control via binding to MBP-GUS (GUS forms dimers) but not to MBP-GTD. (C) Coimmunoprecipitation of the MyoB1-YFP–myosin XI-K complexes formed in vivo. α-GFP:AG, GFP-specific monoclonal antibody immobilized on agarose beads; AG, uncharged agarose beads control. (D) Pull-down assay showing specific binding of the MyoB2 DUF593 domain (dufMyoB2-GFP) to immobilized XI-K (MBP-GTD). Free GFP and N-terminal fragment of MyoB2 (NMyoB2-GFP) provide binding specificity controls. Arrows at the left show protein marker positions with their molecular mass in kilodaltons. Red asterisks mark full-size protein bands; bands of lower molecular mass probably correspond to protein degradation products.

    Techniques Used: Pull Down Assay, Binding Assay, In Vivo, Marker

    18) Product Images from "FORMATION OF FLUORESCENT PROTEINS BY THE ATTACHMENT OF PHYCOERYTHROBILIN TO R-PHYCOERYTHRIN ALPHA AND BETA APO-SUBUNITS"

    Article Title: FORMATION OF FLUORESCENT PROTEINS BY THE ATTACHMENT OF PHYCOERYTHROBILIN TO R-PHYCOERYTHRIN ALPHA AND BETA APO-SUBUNITS

    Journal:

    doi: 10.1016/j.ab.2006.08.011

    Subcellular localization of fusion protein consisting of periplasmic MBP and R-PE apo-alpha subunit after incubation of cells with PEB. Elongated cells were much brighter than normal cells probably because they expressed more of the fusion protein. Fluorescent
    Figure Legend Snippet: Subcellular localization of fusion protein consisting of periplasmic MBP and R-PE apo-alpha subunit after incubation of cells with PEB. Elongated cells were much brighter than normal cells probably because they expressed more of the fusion protein. Fluorescent

    Techniques Used: Incubation

    Photographs comparing E. coli cells expressing periplasmic MBP-R-PE alpha subunit fusion protein and control cells after incubation of cells with PEB chromophore. Vials from left to right contained the following cells incubated with PEB: 1. Control cells
    Figure Legend Snippet: Photographs comparing E. coli cells expressing periplasmic MBP-R-PE alpha subunit fusion protein and control cells after incubation of cells with PEB chromophore. Vials from left to right contained the following cells incubated with PEB: 1. Control cells

    Techniques Used: Expressing, Incubation

    Fluorescence intensities of cells expressing periplasmic MBP-R-PE apo-alpha subunit fusion and control cells after incubation of cells with PEB. Numbers of cells shown on Y-axis were normalized for the purpose of graphical presentation. Fluorescence of
    Figure Legend Snippet: Fluorescence intensities of cells expressing periplasmic MBP-R-PE apo-alpha subunit fusion and control cells after incubation of cells with PEB. Numbers of cells shown on Y-axis were normalized for the purpose of graphical presentation. Fluorescence of

    Techniques Used: Fluorescence, Expressing, Incubation

    19) Product Images from "Mapping the Binding Domain of the F18 Fimbrial Adhesin "

    Article Title: Mapping the Binding Domain of the F18 Fimbrial Adhesin

    Journal:

    doi: 10.1128/IAI.71.4.2163-2172.2003

    SDS-PAGE and Western blot analyses of purified truncated MBP-FedF proteins. Fusion proteins were isolated from E. coli strains carrying pKTH5068, pKTH5069, pKTH5070, pKTH5071, pKTH5072, pKTH5073, pKTH5075, pKTH5076, pKTH5077, or pKTH5094 (lanes 1 to 10, respectively). MBP was used as a control (lane 11). (A) Proteins were run on an SDS-polyacrylamide gel and stained with Coomassie dye. (B) Immunoblots with the corresponding samples using an MBP antiserum.
    Figure Legend Snippet: SDS-PAGE and Western blot analyses of purified truncated MBP-FedF proteins. Fusion proteins were isolated from E. coli strains carrying pKTH5068, pKTH5069, pKTH5070, pKTH5071, pKTH5072, pKTH5073, pKTH5075, pKTH5076, pKTH5077, or pKTH5094 (lanes 1 to 10, respectively). MBP was used as a control (lane 11). (A) Proteins were run on an SDS-polyacrylamide gel and stained with Coomassie dye. (B) Immunoblots with the corresponding samples using an MBP antiserum.

    Techniques Used: SDS Page, Western Blot, Purification, Isolation, Staining

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    Infection:

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Hep2, HeLa or Vero cells grown on coverslips, either transfected or infected, were fixed with 2% formaldehyde/PBS (15 min, room temperature) and permeabilized with 0.5% Triton X-100 (5 min, 4°C). .. Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Mass Spectrometry:

    Article Title: DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation
    Article Snippet: Gels were blotted on nitrocellulose membranes (Bio-Rad) and reacted with anti-MBP monoclonal antibody (New England Biolabs) as described by the manufacturer. .. Gels were blotted on nitrocellulose membranes (Bio-Rad) and reacted with anti-MBP monoclonal antibody (New England Biolabs) as described by the manufacturer.

    Western Blot:

    Article Title: Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda
    Article Snippet: Paragraph title: Western blot analysis ... Anti-MBP Monoclonal antibody (NEB) and Anti-DnaK Monoclonal antibody (Stressgen) were used as periplasmic and cytoplasmic markers.

    Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels
    Article Snippet: For starvation in nutrient-deplete medium, the cells were incubated in Earls Balanced Salt Solution (EBSS; Invitrogen), with the exception of the HEK GFP-DFCP1 cells that were starved as described previously in 140 mM NaCl, 1 mM CaCl2 , 1 mM MgCl2 , 5 mM glucose and 20 mM Hepes, pH 7.4. .. The following primary antibodies were used: mouse anti-cortactin (Upstate, 05-180, 1:1,000), mouse anti-GFP (Clontech, 632381, 1:1,000), mouse anti-Flag (Sigma, F1804, 1:500), mouse anti-MBP (NEB, e8032S, 1:10,000), rabbit anti-ULK1 (Santa Cruz, sc-33182, 1:250), rabbit anti-HS1BP3 (GeneTex, GTX107715, 1:10,000 for WB and 1:500 for IF), rabbit anti-LC3 (Cell Signaling, 27755, 1:1,000 for WB), mouse anti-β-actin (Sigma, SAB1305567 1:20,000), mouse anti-myc (DSHB, 9E10, 1:20), mouse anti-alpha tubulin (Sigma, T5168, 1:20,000), rabbit anti-LC3 (MBL, PM036, 1:500 for IF), mouse anti-p62 (BD biosciences, 610833, 1:1,000 for WB), goat horseradish peroxidase (HRP)-conjugated anti-GST (Abcam, ab58626, 1:10,000 for phosphatidylinositol phosphate (PIP) strips), rabbit anti-phospho-AKT Ser473 (Cell Signaling, 4060, 1:2,000), rabbit anti-phospho-p70-S6K Thr389 (Cell Signaling, 9202, 1:1,000), rabbit anti-p70-S6K (Cell Signaling, 9205, 1:1,000), rabbit anti-ATG16L1 (MBL, PM040, 1:200), mouse anti-TfR CD71 (Santa Cruz, sc-65877, 1:200), mouse anti-WIPI2 (kind gift from Sharon Tooze, 1:2,000), rabbit anti-PLD1 (Cell Signaling, 3832S, 1:200), mouse anti-HA (Abcam, ab18181, 1:200), hamster anti-mAtg9 (kind gift from Sharon Tooze, 1:1,000). .. HRP- and Cy2/3/5-conjugated secondary antibodies were obtained from Jackson Immunolabs.

    Article Title: RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana
    Article Snippet: Finally, six further vigorous washes were performed with the washing buffer (50 mM Tris at pH 7.5, 300 mM NaCl, 0.6% Triton X-100, 1 mM PMSF). .. Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody (NEB E8032S, RRID: AB_1559732 ). .. Long RNA substrates of PHV and At4g29770 were synthesized as described previously ( ).

    Article Title: Cytokinesis requires localized β-actin filament production by an actin isoform specific nucleator
    Article Snippet: The resin was then washed and mixed with 0.02 nmol of different MBP anillin fragments and incubated for 2 h at 4 °C. .. Unbound protein was removed by washing the beads in IB, which were next re-isolated by centrifugation and boiled in SDS sample buffer then analyzed by Western blotting using an anti-MBP monoclonal antibody (E8032, New England Biolabs, 1:2000 dilution) to detect co-purifying anillin fragments. .. To assess the role of anillin and RhoA in regulating the interaction between DIAPH3-NT and DIAPH3-CT, 0.05 nmol of MBP-DIAPH3-NT was immobilized onto 20 μl amylose resin in 100 μl IB as described above and incubated for 1 h at 4 °C.

    Article Title: Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli
    Article Snippet: Immunoblotting was carried out using polyclonal anti-EspB, anti-EspA, anti-Tir, anti-EspC, anti-EspF, anti-EscF, anti-EscI, anti-EscJ, anti-EscUC , or anti-CesT antibodies, as well as anti-DnaK (Assay Designs), horseradish peroxidase (HRP)-conjugated anti-HA (Sigma), HRP-conjugated anti-His (Pierce), or anti-MBP (New England BioLabs) monoclonal antibodies. .. Immunoblotting was carried out using polyclonal anti-EspB, anti-EspA, anti-Tir, anti-EspC, anti-EspF, anti-EscF, anti-EscI, anti-EscJ, anti-EscUC , or anti-CesT antibodies, as well as anti-DnaK (Assay Designs), horseradish peroxidase (HRP)-conjugated anti-HA (Sigma), HRP-conjugated anti-His (Pierce), or anti-MBP (New England BioLabs) monoclonal antibodies.

    Article Title: Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis
    Article Snippet: Harvested cells were crushed by passage through a French press in a solution of 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5% NP-40, 2 mM MgCl2 , benzonase nuclease (10 units; Novagen), and 20% glycerol and centrifuged at 40,000 × g for 30 min. A slurry of affinity resin, either nitrilotriacetic acid-agarose (QIAGEN) charged with Ni2+ or amylose-agarose beads (100 μl; New England Biolabs), was mixed with 500 μl cell extract, and the beads were collected, washed, and then eluted with a solution of 80 μl 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.5% NP-40 containing either 10 mM maltose or 250 mM imidazole. .. Western blotting was performed after electrophoretic transfer of proteins to polyvinylidene difluoride membranes with anti-MBP antiserum (New England Biolabs), His-tagged monoclonal antibody (Novagen), or affinity-purified rabbit polyclonal anti-Rad51 antibodies raised against U. maydis Rad51 protein. .. Complexes were visualized by chemiluminescence with horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences).

    Article Title: Proteome-scale purification of human proteins from bacteria
    Article Snippet: Paragraph title: SDS/PAGE and Western Blot Analysis. ... Antibodies and dilutions were as follows: anti-His4 antibody from Qiagen at 0.1 μl/ml in 3% (wt/vol) BSA; M2 monoclonal anti-FLAG from Pierce at 1:1000 in Blotto (50 mM Tris⋅HCl, pH 7.4/100 mM NaCl/5% nonfat dry milk); Z-5 polyclonal anti-GST from Santa Cruz at 1:1000 in Blotto; and anti-maltose-binding protein (MBP) antibody from NEB at 1:1000 in Blotto.

    Transformation Assay:

    Article Title: RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana
    Article Snippet: All the plasmids were transformed into BL21 competent cells (DE3). .. Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody (NEB E8032S, RRID: AB_1559732 ).

    Article Title: A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex
    Article Snippet: Escherichia coli strain BL21 (Novagen) was transformed with pGEX-3X (Pharmacia), p1704, p2924, and p2925, and induced to express GST, GST-Myo3p-SH3-AD, GST-Myo3p-SH3, and GST-Myo3p-AD, respectively. .. For direct protein–protein interaction experiments, BL21 lysates containing MBP-Vrp1p (1-200) (p3224), MBP-Vrp1p (211-437) (p3349), or MBP-Bee1p (213-222) (p3385) were mixed with purified GST proteins purified from BL21 cells expressing pGEX-3X (Pharmacia), GST-Myo3p-SH3-AD (p1704), or GST-Myo3p-SH3(W1157S)-AD (p1741) and prepared for immunoblot analysis ( ) using monoclonal MBP antibody (New England Biolabs) and monoclonal GST antibody (Santa Cruz).

    Transfection:

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Hep2, HeLa or Vero cells grown on coverslips, either transfected or infected, were fixed with 2% formaldehyde/PBS (15 min, room temperature) and permeabilized with 0.5% Triton X-100 (5 min, 4°C). .. Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Solubility:

    Article Title: Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis
    Article Snippet: E. coli strain BL21(DE3) (Novagen) cells cotransformed with pairs of plasmids expressing proteins with MBP or hexahistidine affinity tags were grown in Luria broth with appropriate antibiotics to mid-log phase, induced with isopropyl-β- d -galactoside, and cultured at 16 or 23°C to maximize solubility of expressed proteins. .. Western blotting was performed after electrophoretic transfer of proteins to polyvinylidene difluoride membranes with anti-MBP antiserum (New England Biolabs), His-tagged monoclonal antibody (Novagen), or affinity-purified rabbit polyclonal anti-Rad51 antibodies raised against U. maydis Rad51 protein.

    Hemagglutination Assay:

    Article Title: A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex
    Article Snippet: GST proteins were purified on glutathione-Sepharose beads, then mixed with yeast extracts (45 min) and prepared for immunoblot analysis ( ) using monoclonal antibody HA.11 (Berkeley Antibody Company), and monoclonal GST antibody (Santa Cruz). .. For direct protein–protein interaction experiments, BL21 lysates containing MBP-Vrp1p (1-200) (p3224), MBP-Vrp1p (211-437) (p3349), or MBP-Bee1p (213-222) (p3385) were mixed with purified GST proteins purified from BL21 cells expressing pGEX-3X (Pharmacia), GST-Myo3p-SH3-AD (p1704), or GST-Myo3p-SH3(W1157S)-AD (p1741) and prepared for immunoblot analysis ( ) using monoclonal MBP antibody (New England Biolabs) and monoclonal GST antibody (Santa Cruz).

    Generated:

    Article Title: A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways
    Article Snippet: Proteins were separated by Tris-glycine SDS-PAGE (7.5%, 10%, 12% or 15% polyacrylamide, as indicated) and transferred onto nitrocellulose membrane either with semi-dry (TransBlot SD SemiDry Transfer Cell, Bio-Rad) or dry transfer (iBlot2, Life technologies). .. Proteins were detected with primary antibodies raised against either Sco2149 (a monoclonal Sco2149 peptide antibody generated by GenScript against the N-Terminal epitope CLPPHEPRVQDVDER), Bla (monoclonal antibody, Abcam ab12251), MBP (monoclonal antibody, NEB E8032L), BamA (polyclonal antibody, [ ]). .. Bands were revealed with chemiluminescence (Clarity Western ECL Blotting Substrate, Biorad) after incubation with secondary antibody coupled to HRP (anti-mouse IgG or Anti-Rabbit IgG, Biorad).

    other:

    Article Title: TC10 controls human myofibril organization and is activated by the sarcomeric RhoGEF obscurin
    Article Snippet: Antibodies were obtained as follow: anti-sarcomeric α-actinin mAb, anti-MHC fast mAb, anti-TC10 pAb, anti-Troponin T mAb and anti-Tubulin mAb from Sigma; anti-Caveolin 3 mAb, anti-Rac mAb, anti-CD56 mAb and anti-Myogenin mAb from BD Transduction Laboratories; anti Cdc42 pAb and anti-HA mAb from Santa cruz Biotechnology, anti-HA pAb from Zymed Laboratories, anti-GFP mAb from Roche, anti-GFP pAb from Torrey Pines Biolabs, anti MBP mAb from New England Biolabs; anti-myc pAb from Medical and Biological Laboratories; anti PAK pAb and anti P PAK pAb from Cell Signaling Technology.

    Article Title: Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments
    Article Snippet: Anti-GST monoclonal antibody was purchased from Rockland Immunochemical (Gilbertsville, PA) and anti-MBP antibody was purchased from New England BioLabs (Ipswich, MA).

    Affinity Purification:

    Article Title: Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis
    Article Snippet: Harvested cells were crushed by passage through a French press in a solution of 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5% NP-40, 2 mM MgCl2 , benzonase nuclease (10 units; Novagen), and 20% glycerol and centrifuged at 40,000 × g for 30 min. A slurry of affinity resin, either nitrilotriacetic acid-agarose (QIAGEN) charged with Ni2+ or amylose-agarose beads (100 μl; New England Biolabs), was mixed with 500 μl cell extract, and the beads were collected, washed, and then eluted with a solution of 80 μl 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.5% NP-40 containing either 10 mM maltose or 250 mM imidazole. .. Western blotting was performed after electrophoretic transfer of proteins to polyvinylidene difluoride membranes with anti-MBP antiserum (New England Biolabs), His-tagged monoclonal antibody (Novagen), or affinity-purified rabbit polyclonal anti-Rad51 antibodies raised against U. maydis Rad51 protein. .. Complexes were visualized by chemiluminescence with horseradish peroxidase-coupled secondary antibodies (Amersham Biosciences).

    Recombinant:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: Purified fusion protein was stored at −80°C until use. .. For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Article Title: The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice
    Article Snippet: To monitor the degradation of the expressed recombinant MBP-Hd1 and MBP proteins, 200 ng purified MBP-Hd1 and MBP protein was added to 75 μL seedling extract for individual assays. .. An equal amount of reactions separated on a 10% SDS-PAGE and detected by immunoblot analysis with anti-MBP antibody (NEB E8032S; 1:10,000 dilution).

    Immunofluorescence:

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Paragraph title: Immunofluorescence microscopy ... Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Pull Down Assay:

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: Paragraph title: In Vitro Pull-Down Assay. ... The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs).

    Flow Cytometry:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Binding of antibodies to the HSV-1 Fc-receptor like proteins gE/gI was blocked with human blood sera of HSV-1 negative individuals/PBS for at least 3 h at room temperature [ ]. .. Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Labeling:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Mouse Assay:

    Article Title: Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii
    Article Snippet: Polyclonal antibodies against TgCdk7, TgCyclinH, TgMat1 and TgRPB1 were raised in mice using following purified GST-TgCdk7, GST-TgCyclinH, His-TgMat1 and His-TgRPB11570-1828 (CTD) proteins respectively. .. The following commercial antibodies were used: His (H1029, Sigma), anti-GFP antibody (ab6556, Abcam), anti-mCherry antibody (PA5-34974, Thermo Scientific), anti-RNAPII CTD repeat P-Ser5 (ab5131, Abcam), anti-RNAPII CTD repeat P-Ser2 (ab5095, Abcam), control IgG (ab46540; Abcam) and anti-m3G-cap & m7G-cap (MABE419, Millipore), anti-MBP antibody (E8032S, New England Biolabs).

    Immunodetection:

    Article Title: Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli
    Article Snippet: Immunoblotting was carried out using polyclonal anti-EspB, anti-EspA, anti-Tir, anti-EspC, anti-EspF, anti-EscF, anti-EscI, anti-EscJ, anti-EscUC , or anti-CesT antibodies, as well as anti-DnaK (Assay Designs), horseradish peroxidase (HRP)-conjugated anti-HA (Sigma), HRP-conjugated anti-His (Pierce), or anti-MBP (New England BioLabs) monoclonal antibodies. .. Immunoblotting was carried out using polyclonal anti-EspB, anti-EspA, anti-Tir, anti-EspC, anti-EspF, anti-EscF, anti-EscI, anti-EscJ, anti-EscUC , or anti-CesT antibodies, as well as anti-DnaK (Assay Designs), horseradish peroxidase (HRP)-conjugated anti-HA (Sigma), HRP-conjugated anti-His (Pierce), or anti-MBP (New England BioLabs) monoclonal antibodies.

    Cell Culture:

    Article Title: Rec2 Interplay with both Brh2 and Rad51 Balances Recombinational Repair in Ustilago maydis
    Article Snippet: E. coli strain BL21(DE3) (Novagen) cells cotransformed with pairs of plasmids expressing proteins with MBP or hexahistidine affinity tags were grown in Luria broth with appropriate antibiotics to mid-log phase, induced with isopropyl-β- d -galactoside, and cultured at 16 or 23°C to maximize solubility of expressed proteins. .. Western blotting was performed after electrophoretic transfer of proteins to polyvinylidene difluoride membranes with anti-MBP antiserum (New England Biolabs), His-tagged monoclonal antibody (Novagen), or affinity-purified rabbit polyclonal anti-Rad51 antibodies raised against U. maydis Rad51 protein.

    Degradation Assay:

    Article Title: The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice
    Article Snippet: Paragraph title: Cell-Free Protein Degradation Assay ... An equal amount of reactions separated on a 10% SDS-PAGE and detected by immunoblot analysis with anti-MBP antibody (NEB E8032S; 1:10,000 dilution).

    Microscopy:

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Paragraph title: Immunofluorescence microscopy ... Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Staining:

    Article Title: DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation
    Article Snippet: Protein purity was determined by staining gels with Code Blue reagent (Pierce). .. Gels were blotted on nitrocellulose membranes (Bio-Rad) and reacted with anti-MBP monoclonal antibody (New England Biolabs) as described by the manufacturer.

    Purification:

    Article Title: A TIR Domain Protein from E. faecalis Attenuates MyD88-Mediated Signaling and NF-κB Activation
    Article Snippet: RAW264.7 macrophages were seeded in 12-well plates, grown to confluence, and then inoculated with 10 µg/ml or 50 µg/ml purified MBP-TcpF or purified MBP control protein for 5 hours at 37°C. .. The membrane was blocked with 5% milk and probed first with anti-MBP monoclonal antibody (New England Biolabs, Ipswich, MA) overnight at 4°C followed by goat anti-mouse IgG conjugated to HRP.

    Article Title: The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice
    Article Snippet: To monitor the degradation of the expressed recombinant MBP-Hd1 and MBP proteins, 200 ng purified MBP-Hd1 and MBP protein was added to 75 μL seedling extract for individual assays. .. An equal amount of reactions separated on a 10% SDS-PAGE and detected by immunoblot analysis with anti-MBP antibody (NEB E8032S; 1:10,000 dilution).

    Article Title: Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii
    Article Snippet: Polyclonal antibodies against TgCdk7, TgCyclinH, TgMat1 and TgRPB1 were raised in mice using following purified GST-TgCdk7, GST-TgCyclinH, His-TgMat1 and His-TgRPB11570-1828 (CTD) proteins respectively. .. The following commercial antibodies were used: His (H1029, Sigma), anti-GFP antibody (ab6556, Abcam), anti-mCherry antibody (PA5-34974, Thermo Scientific), anti-RNAPII CTD repeat P-Ser5 (ab5131, Abcam), anti-RNAPII CTD repeat P-Ser2 (ab5095, Abcam), control IgG (ab46540; Abcam) and anti-m3G-cap & m7G-cap (MABE419, Millipore), anti-MBP antibody (E8032S, New England Biolabs).

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: N-PTM fused to GST protein was purified using glutathione beads (GE Healthcare), and FVE, N-FVE, and C-FVE fused to maltose-binding protein (MBP) were purified using amylose resin (New England Biolabs) according to the manufacturer’s protocol. .. The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs).

    Article Title: A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex
    Article Snippet: GST proteins were purified on glutathione-Sepharose beads, then mixed with yeast extracts (45 min) and prepared for immunoblot analysis ( ) using monoclonal antibody HA.11 (Berkeley Antibody Company), and monoclonal GST antibody (Santa Cruz). .. For direct protein–protein interaction experiments, BL21 lysates containing MBP-Vrp1p (1-200) (p3224), MBP-Vrp1p (211-437) (p3349), or MBP-Bee1p (213-222) (p3385) were mixed with purified GST proteins purified from BL21 cells expressing pGEX-3X (Pharmacia), GST-Myo3p-SH3-AD (p1704), or GST-Myo3p-SH3(W1157S)-AD (p1741) and prepared for immunoblot analysis ( ) using monoclonal MBP antibody (New England Biolabs) and monoclonal GST antibody (Santa Cruz). .. A random peptide 2-hybrid library ( ) was screened with p1190, a pEG202 bait plasmid encoding Myo3p-SH3-AD.

    SDS Page:

    Article Title: The RING-Finger Ubiquitin Ligase HAF1 Mediates Heading date 1 Degradation during Photoperiodic Flowering in Rice
    Article Snippet: The reaction mixtures were incubated at 30°C for the indicated time points. .. An equal amount of reactions separated on a 10% SDS-PAGE and detected by immunoblot analysis with anti-MBP antibody (NEB E8032S; 1:10,000 dilution). .. As a loading control, plant ACTIN abundance was detected with anti-ACTIN antibody (Abmart M20009L;1:1000 dilution).

    Article Title: A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways
    Article Snippet: Proteins were separated by Tris-glycine SDS-PAGE (7.5%, 10%, 12% or 15% polyacrylamide, as indicated) and transferred onto nitrocellulose membrane either with semi-dry (TransBlot SD SemiDry Transfer Cell, Bio-Rad) or dry transfer (iBlot2, Life technologies). .. Proteins were detected with primary antibodies raised against either Sco2149 (a monoclonal Sco2149 peptide antibody generated by GenScript against the N-Terminal epitope CLPPHEPRVQDVDER), Bla (monoclonal antibody, Abcam ab12251), MBP (monoclonal antibody, NEB E8032L), BamA (polyclonal antibody, [ ]).

    Article Title: RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana
    Article Snippet: Finally, six further vigorous washes were performed with the washing buffer (50 mM Tris at pH 7.5, 300 mM NaCl, 0.6% Triton X-100, 1 mM PMSF). .. Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody (NEB E8032S, RRID: AB_1559732 ). .. Long RNA substrates of PHV and At4g29770 were synthesized as described previously ( ).

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: Then 2 µg of GST-N-PTM–bound glutathione beads was incubated with 2 μg of MBP-FVE, MBP-N-FVE, MBP-C-FVE, or MBP alone in binding buffer containing 20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.1% Nonidet P-40 at 4 °C for 2 h. The beads were then washed three times with washing buffer (20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.5% Nonidet P-40). .. The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs). .. EMSA was performed using biotin-labeled probes and the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).

    Article Title: Proteome-scale purification of human proteins from bacteria
    Article Snippet: Paragraph title: SDS/PAGE and Western Blot Analysis. ... Antibodies and dilutions were as follows: anti-His4 antibody from Qiagen at 0.1 μl/ml in 3% (wt/vol) BSA; M2 monoclonal anti-FLAG from Pierce at 1:1000 in Blotto (50 mM Tris⋅HCl, pH 7.4/100 mM NaCl/5% nonfat dry milk); Z-5 polyclonal anti-GST from Santa Cruz at 1:1000 in Blotto; and anti-maltose-binding protein (MBP) antibody from NEB at 1:1000 in Blotto.

    Plasmid Preparation:

    Article Title: Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane
    Article Snippet: A SulfoLink kit was purchased from Pierce. .. A Cholesterol Quantification Kit was from BioVision. pMAL vector, MBP resin, monoclonal anti-MBP antibody, T4 ligase, and restriction enzymes were obtained from New England BioLabs. .. Top Pfu polymerase and BL21 (DE3) bacteria were from Stratagene, reduced-form glutathione and isopropyl β- d -thiogalactopyranoside were from Sigma, proteinase inhibitor mixture set II was from Calbiochem, a DC Protein Assay Kit, SDS/PAGE, and electrophoresis reagents were from Bio-Rad, and a SuperSignal West Pico chemiluminescence detection kit reagent was from Pierce.

    Software:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Article Title: Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli
    Article Snippet: Immunoblotting was carried out using polyclonal anti-EspB, anti-EspA, anti-Tir, anti-EspC, anti-EspF, anti-EscF, anti-EscI, anti-EscJ, anti-EscUC , or anti-CesT antibodies, as well as anti-DnaK (Assay Designs), horseradish peroxidase (HRP)-conjugated anti-HA (Sigma), HRP-conjugated anti-His (Pierce), or anti-MBP (New England BioLabs) monoclonal antibodies. .. For analysis of EscP-HA secretion, the SuperSignal Western Blot Enhancer kit (Pierce) was used before immunodetection.

    Negative Control:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. The extent of recombinant protein binding to beads was analyzed by labeling beads with rabbit polyclonal anti-StrepII tag antibodies (1/40, Genscript, NWSHPQFEK antibody, A00626) and goat anti-rabbit Alexa488 coupled antibodies (Molecular Probes, 1/500) and detecting by flow cytometry in an LSRII (Becton Dickinson) and FLOWJO 8.1 software (Tree Star Inc.).

    Binding Assay:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: Purified fusion protein was stored at −80°C until use. .. For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Article Title: RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana
    Article Snippet: One microgram MBP-fusion Prey proteins of AGO1, AGO10 and AGO10 truncated proteins were preabsorbed for 1 hr at room temperature in 1 mL of binding buffer (50 μl Ni-NTA resin beads, 50 mM Tris at pH 7.5, 150 mM NaCl, 0.2% glycerol, 0.6% Triton X-100, 0.5 mM β-mercaptoethanol, 1 mM PMSF). .. Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody (NEB E8032S, RRID: AB_1559732 ).

    Article Title: Cytokinesis requires localized β-actin filament production by an actin isoform specific nucleator
    Article Snippet: Paragraph title: In vitro binding assays ... Unbound protein was removed by washing the beads in IB, which were next re-isolated by centrifugation and boiled in SDS sample buffer then analyzed by Western blotting using an anti-MBP monoclonal antibody (E8032, New England Biolabs, 1:2000 dilution) to detect co-purifying anillin fragments.

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: Then 2 µg of GST-N-PTM–bound glutathione beads was incubated with 2 μg of MBP-FVE, MBP-N-FVE, MBP-C-FVE, or MBP alone in binding buffer containing 20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.1% Nonidet P-40 at 4 °C for 2 h. The beads were then washed three times with washing buffer (20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.5% Nonidet P-40). .. The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs).

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Binding of antibodies to the HSV-1 Fc-receptor like proteins gE/gI was blocked with human blood sera of HSV-1 negative individuals/PBS for at least 3 h at room temperature [ ]. .. Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    In Vitro:

    Article Title: RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana
    Article Snippet: Paragraph title: In vitro pull-down assays ... Pulled-down proteins were resolved by 6% SDS-PAGE and detected by Western blotting using the α-MBP antibody (NEB E8032S, RRID: AB_1559732 ).

    Article Title: Cytokinesis requires localized β-actin filament production by an actin isoform specific nucleator
    Article Snippet: Paragraph title: In vitro binding assays ... Unbound protein was removed by washing the beads in IB, which were next re-isolated by centrifugation and boiled in SDS sample buffer then analyzed by Western blotting using an anti-MBP monoclonal antibody (E8032, New England Biolabs, 1:2000 dilution) to detect co-purifying anillin fragments.

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: Paragraph title: In Vitro Pull-Down Assay. ... The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs).

    Protein Binding:

    Article Title: DC8 and DC13 var Genes Associated with Severe Malaria Bind Avidly to Diverse Endothelial Cells
    Article Snippet: For the coated bead binding assay, recombinant proteins were coated onto 107 sheep anti-mouse IgG coated Dynal beads (Invitrogen, 110.31) using 1.5 µl of a mouse anti-MBP monoclonal antibody diluted into PBS/0.1% BSA at 1 µg/µl (NEB, E8032S) for 1 hr under rotating agitation. .. After two washes, antibody-coupled beads were incubated with His-MBP-insert-StrepII fusion protein or His-MBP protein for 1–2 hrs, followed by two washes.

    Laser-Scanning Microscopy:

    Article Title: The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain
    Article Snippet: Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J. .. Mouse monoclonal antibodies anti-myc (clone 9E10; kindly provided by J. von Einem), anti-MBP (NEB), anti-ICP0 (Santa Cruz), anti-ICP8 (kindly provided by R. Heilbronn) and anti-VP5 (clone 8F5; kindly provided by J.

    Sampling:

    Article Title: Chloroplast retrograde signal regulates flowering
    Article Snippet: Then 2 µg of GST-N-PTM–bound glutathione beads was incubated with 2 μg of MBP-FVE, MBP-N-FVE, MBP-C-FVE, or MBP alone in binding buffer containing 20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.1% Nonidet P-40 at 4 °C for 2 h. The beads were then washed three times with washing buffer (20 mM Tris⋅Cl pH 7.5, 100 mM NaCl, 5% glycerol, and 0.5% Nonidet P-40). .. The protein eluted from the beads by boiling in 50 µL of 2× sampling buffer was loaded onto the 12.5% SDS/PAGE gel and analyzed by immunoblot assay using anti-MBP antibody (E8032, 1:5,000 dilution; New England Biolabs). .. EMSA was performed using biotin-labeled probes and the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).

    Concentration Assay:

    Article Title: A TIR Domain Protein from E. faecalis Attenuates MyD88-Mediated Signaling and NF-κB Activation
    Article Snippet: Cells were then washed five times with cold PBS and lysates prepared after treatment with trypsin at a final concentration of 0.25% for 5 minutes at 37°C. .. The membrane was blocked with 5% milk and probed first with anti-MBP monoclonal antibody (New England Biolabs, Ipswich, MA) overnight at 4°C followed by goat anti-mouse IgG conjugated to HRP.

    Lysis:

    Article Title: A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex
    Article Snippet: Yeast extracts from strains Y1790 and Y1789 were prepared by growing strains to mid-log in 50 ml culture and then grinding cells with glass beads in lysis buffer (0.1% Triton X-100, 50 mM Tris-HCl, 100 mM NaCl, and 10 mM EDTA). .. For direct protein–protein interaction experiments, BL21 lysates containing MBP-Vrp1p (1-200) (p3224), MBP-Vrp1p (211-437) (p3349), or MBP-Bee1p (213-222) (p3385) were mixed with purified GST proteins purified from BL21 cells expressing pGEX-3X (Pharmacia), GST-Myo3p-SH3-AD (p1704), or GST-Myo3p-SH3(W1157S)-AD (p1741) and prepared for immunoblot analysis ( ) using monoclonal MBP antibody (New England Biolabs) and monoclonal GST antibody (Santa Cruz).

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  • 99
    New England Biolabs mbp
    Effect of <t>Sec16p</t> on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of <t>MBP–Sec16p</t> (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.
    Mbp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs mbp free 25k haga
    Antibodies induced by nasal immunization with <t>25k-hagA-MBP</t> reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.
    Mbp Free 25k Haga, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs 25k haga mbp
    Antibodies induced by nasal immunization with <t>25k-hagA-MBP</t> reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.
    25k Haga Mbp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    25k haga mbp - by Bioz Stars, 2019-12
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    Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Effect of Sec16p on vesicle budding. (A) Microsomal membranes prepared from RSY267 were stripped as described in the legend to Fig. 1 with B88 buffer containing either 0.1 M NaCl or 0.5 M NaCl and centrifuged for 5 min at 10,000 g . Pellets were then resuspended in 100 μl of B88. 10 μl of both pellet (P) and supernatant fractions (S) were separated on 6% SDS-PAGE, transferred to nitrocellulose, and detected with the indicated antibody. (B) Vesicle release (% of total [ 35 S]gpαF released in vesicles) in the presence of saturating amounts of MBP–Sec16p (20 μg/ml) and various amounts of COPII proteins (standard conditions, 1 × COPII: 20 μg/ml Sar1p, 20 μg/ml Sec23/24p, and 50 μg/ml Sec13/31p). Membranes stripped with 0.5 M NaCl were used in budding reactions.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: SDS Page

    Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Effect of MBP–Sec16p and GTP/GMP-PNP on COPII protein recruitment to DOPC/DOPE liposomes. DOPC/DOPE liposomes (corresponding to 25 μg of phospholipids) were incubated with various combinations of COPII proteins, MBP–Sec16p, and nucleotides (48 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP, GTP, or GMP-PNP) for 15 min at 30°C in a 250-μl reaction. Proteins bound to liposomes were recovered by flotation, resolved on SDS-PAGE, and stained with SYPRO red.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Incubation, SDS Page, Staining

    Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Sec16p stimulates COPII vesicle formation from liposomes. (A) Liposomes (corresponding to 12.5 μg phospholipids) were incubated with COPII proteins (80 μg/ml Sar1p, 130 μg/ml Sec23/24p, and 150 μg/ml Sec13/31p), MBP–Sec16p (11 μg/ml, where indicated), and GMP-PNP (100 μM) for 30 min at 27°C, and then sedimented to equilibrium on sucrose density gradients. The fluorescence of Texas red–labeled liposomes in each fraction was measured. (B) Average fluorescence of COPII vesicle peak (fractions 10–12) for three independent gradients (error bars are SEM).

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Incubation, Fluorescence, Labeling

    Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Binding of MBP–Sec16p and COPII proteins to major–minor mix liposomes. (A) Liposomes (corresponding to 25 μg of phospholipids) were incubated with indicated combinations of COPII proteins, MBP–Sec16p, and nucleotides (16 μg/ml Sar1p, 17 μg/ml Sec23/24p, 20 μg/ml Sec13/31p, 10 μg/ml MBP–Sec16p, and 0.1 mM GDP or GMP-PNP) for 15 min at 30°C in 250-μl reactions and then floated on top of a 0.7-M sucrose cushion. Equal amounts of lipids, measured using fluorescent phospholipids ( Matsuoka et al., 1998 ), from floated fractions were applied to 11% SDS-PAGE and stained with SYPRO red. (B) Titration of Sec23/24p. The same amounts of Sar1p, Sec13/31p, MBP–Sec16p, and liposomes as in A were incubated with the indicated amounts of Sec23/24p in the presence of 0.1 mM GMP-PNP, and the binding of proteins was analyzed after liposome flotation. The asterisk indicates a truncated form of Sec31p.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Binding Assay, Incubation, SDS Page, Staining, Titration

    Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Titration of MBP–Sec16p in a liposome binding reaction. (A) Indicated concentrations of MBP–Sec16p were used for supplementation of binding reactions containing major–minor mix liposomes, COPII proteins (16 μg/ml Sar1p, 5 μg/ml Sec23/24p, and 20 μg/ml Sec13/31p), and GMP-PNP (0.1 mM). The asterisk indicates a truncated form of Sec31p. (B) Quantitation of bound proteins shown in A. The amounts of MBP–Sec16p present in 250-μl reactions are listed in the upper right corner of the graph.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Titration, Binding Assay, Quantitation Assay

    Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Overexpression and purification of MBP–Sec16p. (A) Protein composition of salt extracts from ER-enriched microsomes. 100 μg of microsomal membrane proteins from either wild-type FSY3 strain (W) or MBP–Sec16p-overproducing FSY9 strain (O) were incubated on ice in a 100-μl reaction containing 0.5 M NaCl for 15 min. After incubation, mixtures were centrifuged and 10 μl of supernatant fractions were separated on 6% SDS-PAGE and stained with SYPRO red. The left lane contains molecular weight standards (M). (B) Proteins were transferred to nitrocellulose and probed with anti-Sec16p antibody. (C) Salt extract from a 10,000 g membrane pellet was passed through a 6-ml amylose-agarose column and the bound protein was eluted with buffer containing 10 mM maltose. 10 1-ml fractions were collected. 2 μl of salt extract (T), flowthrough (FT), and fractions (E1–10) were separated on 6% SDS-PAGE and stained with SYPRO red stain.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Over Expression, Purification, Incubation, SDS Page, Staining, Molecular Weight

    Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.

    Journal: The Journal of Cell Biology

    Article Title: Sec16p potentiates the action of COPII proteins to bud transport vesicles

    doi: 10.1083/jcb.200207053

    Figure Lengend Snippet: Thin-section electron microscopy of major–minor mix liposomes incubated with and without COPII proteins, GMP-PNP, and MBP–Sec16p. (A) No protein addition showing large, uncoated, uni- and multilamellar liposomes. (B) COPII and GMP-PNP promote coating, budding, and coated vesicle formation. (C) COPII, GMP-PNP, and Sec16p produce groups of vesicular profiles in close apposition to larger liposomes. Filamentous material not seen in B tethers liposomes and coated vesicles together. Bars, 0.2 μm.

    Article Snippet: The expression of the fusion protein was confirmed using antibodies directed against MBP (New England Biolabs, Inc.) and Sec16p.

    Techniques: Electron Microscopy, Incubation

    Antibodies induced by nasal immunization with 25k-hagA-MBP reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Antibodies induced by nasal immunization with 25k-hagA-MBP reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    Comparison of the proportions of CD11c + DCs in various lymphoid tissues. Mice were immunized nasally with 25k-hagA, MBP, or 25k-hagA-MBP as described in the legend to Fig. . DC-enriched cell populations isolated from the spleen,

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Comparison of the proportions of CD11c + DCs in various lymphoid tissues. Mice were immunized nasally with 25k-hagA, MBP, or 25k-hagA-MBP as described in the legend to Fig. . DC-enriched cell populations isolated from the spleen,

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay, Isolation

    Accumulation of 25k-hagA-MBP or CT in neuronal tissues after nasal challenge with acridinium ester-labeled 25k-hagA-MBP (10 μg or 20 μg) or acridinium ester-labeled CT (1 μg or 5 μg). The results are expressed as the mean

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Accumulation of 25k-hagA-MBP or CT in neuronal tissues after nasal challenge with acridinium ester-labeled 25k-hagA-MBP (10 μg or 20 μg) or acridinium ester-labeled CT (1 μg or 5 μg). The results are expressed as the mean

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Labeling

    25k-hagA-specific CD4 + T cell responses in the spleen and CLNs. Groups of mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. . CD4 + T cells were isolated from the spleens or CLNs of nonimmunized

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific CD4 + T cell responses in the spleen and CLNs. Groups of mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. . CD4 + T cells were isolated from the spleens or CLNs of nonimmunized

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay, Isolation

    25k-hagA-specific IgG and IgA antibody responses in serum. Groups of mice were immunized nasally with either 20 μg of 25k-hagA, 20 μg of 25k-hagA-MBP, or 20 μg of 25k-hagA plus 1 μg of CT on days 0, 7, and 14. (A) Time

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific IgG and IgA antibody responses in serum. Groups of mice were immunized nasally with either 20 μg of 25k-hagA, 20 μg of 25k-hagA-MBP, or 20 μg of 25k-hagA plus 1 μg of CT on days 0, 7, and 14. (A) Time

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    25k-hagA-specific IgA antibody response in saliva. Groups of mice were nasally immunized with either 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT as described in the legend to Fig. . (A) Saliva samples were collected 7 days after the final

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific IgA antibody response in saliva. Groups of mice were nasally immunized with either 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT as described in the legend to Fig. . (A) Saliva samples were collected 7 days after the final

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses in TLR4 −/− mice. Groups of TLR4 −/− or TLR4 +/+ mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses in TLR4 −/− mice. Groups of TLR4 −/− or TLR4 +/+ mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques:

    Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques:

    Antibodies induced by nasal immunization with 25k-hagA-MBP reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Antibodies induced by nasal immunization with 25k-hagA-MBP reduced P. gingivalis -induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    Comparison of the proportions of CD11c + DCs in various lymphoid tissues. Mice were immunized nasally with 25k-hagA, MBP, or 25k-hagA-MBP as described in the legend to Fig. . DC-enriched cell populations isolated from the spleen,

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Comparison of the proportions of CD11c + DCs in various lymphoid tissues. Mice were immunized nasally with 25k-hagA, MBP, or 25k-hagA-MBP as described in the legend to Fig. . DC-enriched cell populations isolated from the spleen,

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay, Isolation

    Accumulation of 25k-hagA-MBP or CT in neuronal tissues after nasal challenge with acridinium ester-labeled 25k-hagA-MBP (10 μg or 20 μg) or acridinium ester-labeled CT (1 μg or 5 μg). The results are expressed as the mean

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Accumulation of 25k-hagA-MBP or CT in neuronal tissues after nasal challenge with acridinium ester-labeled 25k-hagA-MBP (10 μg or 20 μg) or acridinium ester-labeled CT (1 μg or 5 μg). The results are expressed as the mean

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Labeling

    25k-hagA-specific CD4 + T cell responses in the spleen and CLNs. Groups of mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. . CD4 + T cells were isolated from the spleens or CLNs of nonimmunized

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific CD4 + T cell responses in the spleen and CLNs. Groups of mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. . CD4 + T cells were isolated from the spleens or CLNs of nonimmunized

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay, Isolation

    25k-hagA-specific IgG and IgA antibody responses in serum. Groups of mice were immunized nasally with either 20 μg of 25k-hagA, 20 μg of 25k-hagA-MBP, or 20 μg of 25k-hagA plus 1 μg of CT on days 0, 7, and 14. (A) Time

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific IgG and IgA antibody responses in serum. Groups of mice were immunized nasally with either 20 μg of 25k-hagA, 20 μg of 25k-hagA-MBP, or 20 μg of 25k-hagA plus 1 μg of CT on days 0, 7, and 14. (A) Time

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    25k-hagA-specific IgA antibody response in saliva. Groups of mice were nasally immunized with either 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT as described in the legend to Fig. . (A) Saliva samples were collected 7 days after the final

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific IgA antibody response in saliva. Groups of mice were nasally immunized with either 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT as described in the legend to Fig. . (A) Saliva samples were collected 7 days after the final

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses in TLR4 −/− mice. Groups of TLR4 −/− or TLR4 +/+ mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: 25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses in TLR4 −/− mice. Groups of TLR4 −/− or TLR4 +/+ mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: Mouse Assay

    Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques:

    Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Journal:

    Article Title: Nasal Immunization with a Fusion Protein Consisting of the Hemagglutinin A Antigenic Region and the Maltose-Binding Protein Elicits CD11c+ CD8+ Dendritic Cells for Induced Long-Term Protective Immunity

    doi: 10.1128/IAI.01203-10

    Figure Lengend Snippet: Nasal administration of 25k-hagA-MBP expands CD11c+ CD8α+ DCs in mucosal and systemic lymphoid tissues.

    Article Snippet: 25k-hagA-MBP and MBP-free 25k-hagA were purified to homogeneity utilizing amylase resin affinity chromatography (New England Biolabs).

    Techniques: