Structured Review

Jackson Immuno hrp conjugated anti rabbit igg
Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or <t>IgG</t> as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as <t>streptavidin-HRP.</t>
Hrp Conjugated Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hrp conjugated anti rabbit igg/product/Jackson Immuno
Average 90 stars, based on 6 article reviews
Price from $9.99 to $1999.99
hrp conjugated anti rabbit igg - by Bioz Stars, 2020-08
90/100 stars

Images

1) Product Images from "Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis"

Article Title: Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx326

Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.
Figure Legend Snippet: Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.

Techniques Used: Mass Spectrometry, Binding Assay, Incubation, Immunoprecipitation

2) Product Images from "The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells"

Article Title: The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-32200-z

( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.
Figure Legend Snippet: ( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.

Techniques Used: Incubation, Serial Dilution, Purification, Activity Assay, Western Blot

3) Product Images from "The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells"

Article Title: The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-32200-z

( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.
Figure Legend Snippet: ( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.

Techniques Used: Incubation, Serial Dilution, Purification, Activity Assay, Western Blot

4) Product Images from "Characterization of circulating APOL1 protein complexes in African Americans [S]"

Article Title: Characterization of circulating APOL1 protein complexes in African Americans [S]

Journal: Journal of Lipid Research

doi: 10.1194/jlr.M063453

Confirmation of APOL1-associated protein components. A: Co-immunoprecipitation of APOL1 with protein components in whole serum using the N-terminal rabbit anti-APOL1 antibody (APOL1) and pre-bleed normal IgG (Pre-bleed). APOL1, APOA1, and HPR are confirmed
Figure Legend Snippet: Confirmation of APOL1-associated protein components. A: Co-immunoprecipitation of APOL1 with protein components in whole serum using the N-terminal rabbit anti-APOL1 antibody (APOL1) and pre-bleed normal IgG (Pre-bleed). APOL1, APOA1, and HPR are confirmed

Techniques Used: Immunoprecipitation

Related Articles

Incubation:

Article Title: The maintenance ability and Ca2+ availability of skeletal muscle are enhanced by sildenafil
Article Snippet: .. The proteins on the gel were transferred to a polyvinylidene difluoride membrane at 100 V for 1 h. The membranes were blocked with 5% (w/v) non-fat milk dissolved in PBS, incubated with a corresponding primary antibody, washed three times with PBS containing 0.1% Tween20 and then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-goat (205-035-108), anti-mouse (715-035-151), anti-rabbit (711-035-152), 1:50 000 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature (24 °C). .. The membranes were washed three times with PBS and developed using a SuperSignal Ultra Chemiluminescent substrate (Pierce, Rockford, IL, USA).

Article Title: Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors
Article Snippet: .. After five washes in PBS-Tween 0.5%, the membrane was incubated with a peroxidase-conjugated secondary antibody (Jackson Immunoresearch, catalog no. 711-035-152) diluted 1:5000 for 1 h at RT. .. Signal was detected by enhanced chemiluminescence (ECL, Amersham Biosciences, GE Healthcare, catalog no. W9643350) using a GeneGnome HR System (Syngene).

other:

Article Title: Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control
Article Snippet: Secondary antibodies used were HRP-conjugated donkey anti-mouse (1:5,000, Jackson ImmunoResearch catalogue number 715-035-150), donkey anti-rabbit (1:5,000, Jackson ImmunoResearch catalogue number 711-035-152) or donkey anti-human (1:5,000, Jackson ImmunoResearch catalogue number 709-035-149).

Article Title: Regulation of Krüppel-like factor 8 by the NEDD4 E3 ubiquitin ligase
Article Snippet: Secondary antibodies were HRP-conjugated donkey anti-mouse (715-035-150) and donkey anti-rabbit IgG (711-035-152) from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Article Title: Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes
Article Snippet: Horseradish peroxidase (HRP)-conjugated antibodies: donkey anti-mouse immunoglobulin G (IgG) (H+L; Jackson ImmunoResearch Labs; 715-035-150), donkey anti-rabbit IgG (H+L; Jackson ImmunoResearch Labs; 711-035-152), and mouse anti-GAPDH (1:10,000, Sigma Aldrich; 45-9295).

Article Title: The PSMA8 subunit of the spermatoproteasome is essential for proper meiotic exit and mouse fertility
Article Snippet: Secondary horseradish peroxidase-conjugated α-mouse (715-035-150, Jackson ImmunoResearch), α-rabbit (711-035-152, Jackson ImmunoResearch), or α-goat (705-035-147, Jackson ImmunoResearch) antibodies were used at 1:5000 dilution.

Article Title: CD98hc (SLC3A2) sustains amino acid and nucleotide availability for cell cycle progression
Article Snippet: Immunoreactive bands were detected with horseradish peroxidase anti-mouse (715-035-150, Jackson Immuno Research Europe, 1:10.000), anti-rabbit (711-035-152, Jackson Immuno Research Europe, 1:10.000), or anti-rat (SC-2956, Santa Cruz, 1:15.000) antibodies using the ECL system (RPN2106, Sigma-Aldrich).

Article Title: Obesity, Diabetes and Energy Homeostasis: Heat shock protein 72 regulates hepatic lipid accumulation
Article Snippet: Secondary antibodies used included goat anti-mouse (cat. no. 170-5047, Bio-Rad), donkey anti-rabbit (cat. no. 711-035-152, Jackson ImmunoResearch, West Grove, PA), and goat anti-rabbit (cat. no. sc-2004, Santa Cruz Biotechnology, Dallas, TX).

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    Jackson Immuno hrp conjugated anti rabbit igg
    Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or <t>IgG</t> as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as <t>streptavidin-HRP.</t>
    Hrp Conjugated Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated anti rabbit igg/product/Jackson Immuno
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated anti rabbit igg - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Jackson Immuno hrp conjugated goat anti rabbit igg
    The eLtaS-specific monoclonal antibody MAE4 efficiently prevents S. aureus infection. ( a ) The binding affinity of MAE4 to eLtaS was determined by ELISA. eLtaS protein was coated onto 96-well plates in the presence of a serial dilution of MAE4. Bound <t>IgG</t> was detected using peroxidase <t>(HRP)-conjugated</t> goat anti-mouse IgG. ( b,c ) Assessment of the anti-infective effect of MAE4 in a murine model of acute peritoneal infection. MAE4 (100 μg/mouse) was injected into the peritoneal cavity 2 h prior to challenge with S. aureus 8325-4 (5 × 10 8 cfu/mouse) ( b ) or Δ ltaS (6 × 10 9 cfu/mouse) ( c ). The survival rate was measured at different time points post challenge. Data are presented as the percentage of mice surviving. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8). ( d ) MAE4 protects mice from S. aureus infection in a pneumonia infection model. CD-1 mice were injected intratracheally with S. aureus 8325-4 cells (1 × 10 7 cfu/mouse). MAE4 IgG (100 μg/mouse) was injected intramuscularly into the left hind leg at 30 min, 24 h, and 48 h post infection. Lung sections were taken 72 h post challenge and stained with hematoxylin-eosin. ( e–g ) Determination of the effect of MAE4 in a sublethal murine model of intravenous challenge. MAE4 IgG (100 μg/mouse) was injected into the peritoneal cavity of BALB/c mice 2 h prior to intravenous challenge with S. aureus Newman (1 × 10 7 cfu/mouse). Kidneys were examined by histopathology for internal abscesses 5 days post infection. Staphylococcal abscess (black arrow) with a central concentration of staphylococci (blue arrow) is indicated ( e ). Bacterial colonies were counted at 5 days post infection ( f ). Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student’s t test. For the lethal challenge model, S. aureus Newman cells (1 × 10 8 cfu/mouse) were administered and survival rates were monitored for 10 days ( g ). Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8) (NS, non-significant).
    Hrp Conjugated Goat Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated goat anti rabbit igg/product/Jackson Immuno
    Average 92 stars, based on 111 article reviews
    Price from $9.99 to $1999.99
    hrp conjugated goat anti rabbit igg - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.

    Journal: Nucleic Acids Research

    Article Title: Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis

    doi: 10.1093/nar/gkx326

    Figure Lengend Snippet: Association of hnRNP C with polysomes increases from G1 to mitosis. ( A ) MS measurements of members of the hnRNP family. Bar plot represents the logarithmic ratio of normalized MS intensities for M over G1; heatmaps reflect the relative abundance of each protein during mitosis in either ribosome-associated (‘Ribosome’) or total proteome (‘Total’) samples. ( B ) Polysomes were extracted from G1 and M cytoplasmic lysates by ultracentrifugation through a sucrose cushion, and protein content of the lysates and pellets was monitored by immunoblotting using antibodies to hnRNP C, Poly-A binding protein (PABPC1) and Ribosomal protein L26 (RPL26). ( C ) Polysomes were extracted from G1 and M cells by ultracentrifugation through a sucrose gradient, and protein content of each fraction was monitored by immunoblotting using antibodies to Ribosomal protein P0 (RPLP0) and either hnRNP C (top panel) or SRSF1 (bottom panel). The membrane was simultaneously incubated with either pair of antibodies to allow a more direct comparison of protein amounts. 40S, small ribosomal subunit. ( D ) Polysomes were extracted as in (A) and hnRNP C-bound complexes were immunoprecipitated using antibodies to hnRNP C or IgG as control, followed by incubation with biotin-conjugated puromycin to label nascent polypeptide chains. 5% of polysome pellet and 100% of hnRNP C IP were then subjected to immunoblot analysis using anti-hnRNP C and anti-RPL26, as well as streptavidin-HRP.

    Article Snippet: Secondary antibodies: HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, (Jackson ImmunoResearch Laboratories, 1:10 000).

    Techniques: Mass Spectrometry, Binding Assay, Incubation, Immunoprecipitation

    ( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.

    Journal: Scientific Reports

    Article Title: The Fab fragment of anti-IgE Cε2 domain prevents allergic reactions through interacting with IgE-FcεRIα complex on rat mast cells

    doi: 10.1038/s41598-018-32200-z

    Figure Lengend Snippet: ( a ) Fab-6HD5 inhibits mast cell degranulation. RBL-2H3 cells were sensitized overnight with IgE (SPE-7) and further incubated with a serial dilution of highly purified Fab-6HD5 (2–20 μg/ml) for 2 hours at 37 °C. After washing, cells were incubated with DNP-BSA for 1 hour at 37 °C. The supernatant was then incubated with p-nitrophenyl N-acetyl-beta-D-glucosamine (PNAG) for 1 hour at 4 °C. IgE-mediated degranulation was monitored by β-hexosaminidase activity. The amount of β-hexosaminidase was determined by measuring the optical density at 405 nm. ( b ) Fab-6HD5 inhibits Syk phosphorylation. RBL-2H3 cells were incubated with anti-TNP IgE (0.5 μg/ml) for 1 hour. Cells were further incubated with highly purified 6HD5-Fab (2 μg/ml) or control IgG2a overnight. After washing, cells were stimulated with TNP26-BSA (100 ng/ml) for indicated periods and cell extracts were subjected to Western blotting. Proteins were detected with anti-phosphorylated Syk, anti-Syk, and anti-Tpt1 antibodies followed by HRP-conjugated anti-rabbit or anti-mouse antibody.

    Article Snippet: After washing the membranes three times with 0.05% Tween-PBS and blocking them with BlockAce (DS Pharma Biomedical), the membranes were re-probed with anti-GST-tag pAb (MBL, 1:1000) followed by HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch, 1:20000).

    Techniques: Incubation, Serial Dilution, Purification, Activity Assay, Western Blot

    The eLtaS-specific monoclonal antibody MAE4 efficiently prevents S. aureus infection. ( a ) The binding affinity of MAE4 to eLtaS was determined by ELISA. eLtaS protein was coated onto 96-well plates in the presence of a serial dilution of MAE4. Bound IgG was detected using peroxidase (HRP)-conjugated goat anti-mouse IgG. ( b,c ) Assessment of the anti-infective effect of MAE4 in a murine model of acute peritoneal infection. MAE4 (100 μg/mouse) was injected into the peritoneal cavity 2 h prior to challenge with S. aureus 8325-4 (5 × 10 8 cfu/mouse) ( b ) or Δ ltaS (6 × 10 9 cfu/mouse) ( c ). The survival rate was measured at different time points post challenge. Data are presented as the percentage of mice surviving. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8). ( d ) MAE4 protects mice from S. aureus infection in a pneumonia infection model. CD-1 mice were injected intratracheally with S. aureus 8325-4 cells (1 × 10 7 cfu/mouse). MAE4 IgG (100 μg/mouse) was injected intramuscularly into the left hind leg at 30 min, 24 h, and 48 h post infection. Lung sections were taken 72 h post challenge and stained with hematoxylin-eosin. ( e–g ) Determination of the effect of MAE4 in a sublethal murine model of intravenous challenge. MAE4 IgG (100 μg/mouse) was injected into the peritoneal cavity of BALB/c mice 2 h prior to intravenous challenge with S. aureus Newman (1 × 10 7 cfu/mouse). Kidneys were examined by histopathology for internal abscesses 5 days post infection. Staphylococcal abscess (black arrow) with a central concentration of staphylococci (blue arrow) is indicated ( e ). Bacterial colonies were counted at 5 days post infection ( f ). Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student’s t test. For the lethal challenge model, S. aureus Newman cells (1 × 10 8 cfu/mouse) were administered and survival rates were monitored for 10 days ( g ). Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8) (NS, non-significant).

    Journal: Scientific Reports

    Article Title: MAE4, an eLtaS monoclonal antibody, blocks Staphylococcus aureus virulence

    doi: 10.1038/srep17215

    Figure Lengend Snippet: The eLtaS-specific monoclonal antibody MAE4 efficiently prevents S. aureus infection. ( a ) The binding affinity of MAE4 to eLtaS was determined by ELISA. eLtaS protein was coated onto 96-well plates in the presence of a serial dilution of MAE4. Bound IgG was detected using peroxidase (HRP)-conjugated goat anti-mouse IgG. ( b,c ) Assessment of the anti-infective effect of MAE4 in a murine model of acute peritoneal infection. MAE4 (100 μg/mouse) was injected into the peritoneal cavity 2 h prior to challenge with S. aureus 8325-4 (5 × 10 8 cfu/mouse) ( b ) or Δ ltaS (6 × 10 9 cfu/mouse) ( c ). The survival rate was measured at different time points post challenge. Data are presented as the percentage of mice surviving. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8). ( d ) MAE4 protects mice from S. aureus infection in a pneumonia infection model. CD-1 mice were injected intratracheally with S. aureus 8325-4 cells (1 × 10 7 cfu/mouse). MAE4 IgG (100 μg/mouse) was injected intramuscularly into the left hind leg at 30 min, 24 h, and 48 h post infection. Lung sections were taken 72 h post challenge and stained with hematoxylin-eosin. ( e–g ) Determination of the effect of MAE4 in a sublethal murine model of intravenous challenge. MAE4 IgG (100 μg/mouse) was injected into the peritoneal cavity of BALB/c mice 2 h prior to intravenous challenge with S. aureus Newman (1 × 10 7 cfu/mouse). Kidneys were examined by histopathology for internal abscesses 5 days post infection. Staphylococcal abscess (black arrow) with a central concentration of staphylococci (blue arrow) is indicated ( e ). Bacterial colonies were counted at 5 days post infection ( f ). Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student’s t test. For the lethal challenge model, S. aureus Newman cells (1 × 10 8 cfu/mouse) were administered and survival rates were monitored for 10 days ( g ). Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8) (NS, non-significant).

    Article Snippet: Bound C3b proteins were detected using anti-C3 (Santa Cruz Biotechnology) followed by HRP-conjugated goat anti-rabbit IgG (1:20,000, Jackson ImmunoResearch Laboratories).

    Techniques: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Serial Dilution, Injection, Mouse Assay, Staining, Histopathology, Concentration Assay, Two Tailed Test

    Identification of OTUD4 as a MAVS-interacting DUB. a Immunoprecipitation (IP, with anti-FLAG) and immunoblot (IB, with anti-FLAG and anti-HA and goat anti-mouse IgG(H+L) antibody) analysis of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged DUBs for 24 h. Cell lysates were analyzed by immunoblot with anti-FLAG or anti-HA. b Immunoprecipitation (with control IgG or anti-OTUD4) and immunoblot (with anti-OTUD4 and anti-MAVS and goat anti-mouse IgG F(ab’)2 fragment specific antibody) of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with SeV or VSV for 5–10 h. Cell lysates were analyzed by immunoblot with antibodies against the indicated proteins. The graphs show relative intensities of OTUD4 obtained by normalizing the intensities of OTUD4 to the intensities of β-Actin. c , d Immunoblot of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged OTUD4 or mutants ( c ) or with plasmids encoding GFP-OTUD4 and FLAG-tagged MAVS or truncates ( d ), lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-FLAG-HRP, anti-GFP or anti-HA. Data are representative of three ( a ) or two ( b–d ) independent experiments (mean ± S.D. in b )

    Journal: Cell Research

    Article Title: Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS

    doi: 10.1038/s41422-018-0107-6

    Figure Lengend Snippet: Identification of OTUD4 as a MAVS-interacting DUB. a Immunoprecipitation (IP, with anti-FLAG) and immunoblot (IB, with anti-FLAG and anti-HA and goat anti-mouse IgG(H+L) antibody) analysis of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged DUBs for 24 h. Cell lysates were analyzed by immunoblot with anti-FLAG or anti-HA. b Immunoprecipitation (with control IgG or anti-OTUD4) and immunoblot (with anti-OTUD4 and anti-MAVS and goat anti-mouse IgG F(ab’)2 fragment specific antibody) of MEFs (upper panels) and BMDCs (lower panels) that were left uninfected or infected with SeV or VSV for 5–10 h. Cell lysates were analyzed by immunoblot with antibodies against the indicated proteins. The graphs show relative intensities of OTUD4 obtained by normalizing the intensities of OTUD4 to the intensities of β-Actin. c , d Immunoblot of HEK293 cells that were transfected with plasmids encoding HA-MAVS and FLAG-tagged OTUD4 or mutants ( c ) or with plasmids encoding GFP-OTUD4 and FLAG-tagged MAVS or truncates ( d ), lysed and immunoprecipitated with anti-FLAG. Cell lysate was analyzed by immunoblot with anti-FLAG or anti-FLAG-HRP, anti-GFP or anti-HA. Data are representative of three ( a ) or two ( b–d ) independent experiments (mean ± S.D. in b )

    Article Snippet: Immuno-reagents were obtained as follows: Mouse control IgG (Santa Cruz Biotechnology, sc-2025); rabbit control IgG (Millipore, 12–370); HRP-conjugated goat-anti mouse or rabbit IgG (Thermo Scientific, PA1-86717 and SA1-9510); HRP-conjugated mouse anti-FLAG (Sigma, A8592); HRP-conjugated goat anti-mouse IgG, F(ab′)2 fragment specific (Jackson Immuno Research,115-035-006); HRP-conjugated goat anti-rabbit IgG, F(ab′)2 fragment specific (Jackson Immuno Research,111-035-006); mouse anti-FLAG (Sungene, KM8002); anti-GFP (Sungene, KM8009); anti-β-Actin (KM9001); anti-GAPDH (KM9002); anti-Myc (KM8003); anti-Tubulin (KM9003); anti-HA (COVANCE, MMS-101R); anti-Ubiquitin (sc-8017); anti-pIκBα (Cell Singling Technologies, 9246L); anti-mouse MAVS (sc-365333) and anti-IRF3 (sc-33641); Rabbit anti-ubiquitin K48-specific linkage (Millipore, 05-1307); anti-ubiquitin K63-specific linkage (Millipore, 05-1308); anti-TBK1 (Abcam, 96328-11); anti-p-TBK1 (Abcam, 109272), anti-IRF3 (sc-9082); anti-p-IRF3 (Cell Singling Technologies, 4947S); anti-IκBα (sc-371); anti-mouse MAVS (CST 4983s) and anti-human MAVS (BETHYL, A300-782A).

    Techniques: Immunoprecipitation, Transfection, Infection