flag lysis buffer  (Roche)


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    Structured Review

    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
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    Images

    1) Product Images from "Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation"

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    Journal: Journal of viral hepatitis

    doi: 10.1111/jvh.12225

    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Figure Legend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Techniques Used: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    2) Product Images from "Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact"

    Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036349

    Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.
    Figure Legend Snippet: Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.

    Techniques Used:

    Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.
    Figure Legend Snippet: Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.

    Techniques Used: In Vivo, In Vitro, Planar Chromatography, Polymerase Chain Reaction, Transfection

    3) Product Images from "Analytical biochemistry of DNA-protein assemblies from crude cell extracts"

    Article Title: Analytical biochemistry of DNA-protein assemblies from crude cell extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm490

    Affinity purification of the DNA ends-binding proteins. The proteins were purified from HeLa nuclear extracts using the PCB-TetO target sequence immobilized on beads (lanes 2 and 4) and without oligonucleotide on beads as control (lane 3). ( A ) The chromatographic slurry was either treated with detergents (lane 2) or irradiated (lanes 3 and 4), then the released proteins were separated on an 8% SDS-PAGE gel and stained with Sypro Ruby protein gel stain. ( B ) The purification product obtained after irradiation was analyzed by western blot with antibodies against the DNA ends-binding proteins as indicated. MW, molecular weight markers.
    Figure Legend Snippet: Affinity purification of the DNA ends-binding proteins. The proteins were purified from HeLa nuclear extracts using the PCB-TetO target sequence immobilized on beads (lanes 2 and 4) and without oligonucleotide on beads as control (lane 3). ( A ) The chromatographic slurry was either treated with detergents (lane 2) or irradiated (lanes 3 and 4), then the released proteins were separated on an 8% SDS-PAGE gel and stained with Sypro Ruby protein gel stain. ( B ) The purification product obtained after irradiation was analyzed by western blot with antibodies against the DNA ends-binding proteins as indicated. MW, molecular weight markers.

    Techniques Used: Affinity Purification, Binding Assay, Purification, Sequencing, Irradiation, SDS Page, Staining, Western Blot, Molecular Weight

    Oligonucleotide ligation by DNA ends-binding protein complexes. After washes, the DNA–protein assemblies were incubated with ATP to allow the ligation of the TetO sequence. In lane 3, the ligation was performed on the beads in presence of ATP then the nucleic sequence was released after irradiation of the chromatographic slurry. In lane 4, the DNA–protein assemblies were recovered after the irradiation step then incubated with ATP. The oligonucleotides were resolved on an 8% non-denaturing polyacrylamide gel and revealed by Southern blot with the radiolabeled non-photocleavable strand of the PCB-TetO duplex. Lane 1 shows the intact TetO oligonucleotide and lane 2 the oligonucleotide ligated by the T4 DNA ligase protein.
    Figure Legend Snippet: Oligonucleotide ligation by DNA ends-binding protein complexes. After washes, the DNA–protein assemblies were incubated with ATP to allow the ligation of the TetO sequence. In lane 3, the ligation was performed on the beads in presence of ATP then the nucleic sequence was released after irradiation of the chromatographic slurry. In lane 4, the DNA–protein assemblies were recovered after the irradiation step then incubated with ATP. The oligonucleotides were resolved on an 8% non-denaturing polyacrylamide gel and revealed by Southern blot with the radiolabeled non-photocleavable strand of the PCB-TetO duplex. Lane 1 shows the intact TetO oligonucleotide and lane 2 the oligonucleotide ligated by the T4 DNA ligase protein.

    Techniques Used: Ligation, Binding Assay, Incubation, Sequencing, Irradiation, Southern Blot

    EMSA analysis of fractions obtained from nucleic acid-binding protein purification. ( A ) To observe the band shift corresponding to the TetR-TetO interaction, whole cell extracts were mixed with the PCB-TetO bait in absence or in presence of tetracycline (Tet; lanes 2 and 3, respectively). To perform the purification, whole cell extract was incubated with the PCB-TetO immobilized on beads, then the presence of the complex was analyzed in the purification product (lane 4). ( B ) Tetracycline was mixed with the purification product to confirm the specificity of the DNA–protein interaction. The asterisk indicates that only 1/2 of the corresponding fraction was loaded.
    Figure Legend Snippet: EMSA analysis of fractions obtained from nucleic acid-binding protein purification. ( A ) To observe the band shift corresponding to the TetR-TetO interaction, whole cell extracts were mixed with the PCB-TetO bait in absence or in presence of tetracycline (Tet; lanes 2 and 3, respectively). To perform the purification, whole cell extract was incubated with the PCB-TetO immobilized on beads, then the presence of the complex was analyzed in the purification product (lane 4). ( B ) Tetracycline was mixed with the purification product to confirm the specificity of the DNA–protein interaction. The asterisk indicates that only 1/2 of the corresponding fraction was loaded.

    Techniques Used: Binding Assay, Protein Purification, Electrophoretic Mobility Shift Assay, Purification, Incubation

    4) Product Images from "IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma"

    Article Title: IL-17 Enhances Chemotaxis of Primary Human B Cells during Asthma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114604

    Th-17 cytokines enhance B cell migration in vitro. (A) Migration of asthmatic and healthy B cells towards a gradient of IL-17A, IL-17F, or IL-17A+F in vitro (n = 6). (B) Representative picture of B cells migrating towards IL-17A cytokine. Data is presented as % migration of B cells relative to negative control (medium). Data is expressed as means ± SD. *P
    Figure Legend Snippet: Th-17 cytokines enhance B cell migration in vitro. (A) Migration of asthmatic and healthy B cells towards a gradient of IL-17A, IL-17F, or IL-17A+F in vitro (n = 6). (B) Representative picture of B cells migrating towards IL-17A cytokine. Data is presented as % migration of B cells relative to negative control (medium). Data is expressed as means ± SD. *P

    Techniques Used: Migration, In Vitro, Negative Control

    5) Product Images from "Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells"

    Article Title: Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells

    Journal: Nature Communications

    doi: 10.1038/ncomms12526

    O -GlcNAcylation accumulation as a result of TG treatment promotes B-cell activation and apoptosis. ( a ) Immunoblotting (IB) showing the expression of OGA in mouse primary splenic B cells after anti-IgM (10 μg ml −1 ) stimulation at various time points. ( b ) IB showing the levels of O -GlcNAcylation after anti-IgM (10 μg ml −1 ) stimulation. ( c ) IB showing elevated levels of O -GlcNAcylated proteins after TG (1.0 μM) treatment of mouse splenic B cells. The specificity of anti- O -GlcNAc is validated in the presence of a 0.5 M GlcNAc competitor. ( d ) Flow cytometric analysis of surface CD86 expression showing the effect of pretreatment with TG (1.0 μM) for 8 h on the activation of mouse splenic B cells by anti-IgM (0.5 μg ml −1 ) treatment. ( e ) Levels of Ca 2+ influx measured by Fluo-4 labelling after pretreatment with TG (1.0 μM) for 8 h and stimulation with indicated doses of anti-IgM. ( f ) IB showing increased levels of Lyn phosphorylation on tyrosine 397 in mouse splenic B cells that were pretreated with TG and stimulated with anti-IgM (10 μg ml −1 ) at the indicated time points. ( g ) Cell viability determined by trypan blue staining of splenic B cells that were pretreated with TG (shown as grey bar) and stimulated with anti-IgM (10 μg ml −1 ) for 24, 48 and 72 h. Results represent the mean±s.e.m. ( n =3). * P
    Figure Legend Snippet: O -GlcNAcylation accumulation as a result of TG treatment promotes B-cell activation and apoptosis. ( a ) Immunoblotting (IB) showing the expression of OGA in mouse primary splenic B cells after anti-IgM (10 μg ml −1 ) stimulation at various time points. ( b ) IB showing the levels of O -GlcNAcylation after anti-IgM (10 μg ml −1 ) stimulation. ( c ) IB showing elevated levels of O -GlcNAcylated proteins after TG (1.0 μM) treatment of mouse splenic B cells. The specificity of anti- O -GlcNAc is validated in the presence of a 0.5 M GlcNAc competitor. ( d ) Flow cytometric analysis of surface CD86 expression showing the effect of pretreatment with TG (1.0 μM) for 8 h on the activation of mouse splenic B cells by anti-IgM (0.5 μg ml −1 ) treatment. ( e ) Levels of Ca 2+ influx measured by Fluo-4 labelling after pretreatment with TG (1.0 μM) for 8 h and stimulation with indicated doses of anti-IgM. ( f ) IB showing increased levels of Lyn phosphorylation on tyrosine 397 in mouse splenic B cells that were pretreated with TG and stimulated with anti-IgM (10 μg ml −1 ) at the indicated time points. ( g ) Cell viability determined by trypan blue staining of splenic B cells that were pretreated with TG (shown as grey bar) and stimulated with anti-IgM (10 μg ml −1 ) for 24, 48 and 72 h. Results represent the mean±s.e.m. ( n =3). * P

    Techniques Used: Activation Assay, Expressing, Flow Cytometry, Staining

    Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by immunoblotting (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit IgG (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P
    Figure Legend Snippet: Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by immunoblotting (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit IgG (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P

    Techniques Used: Mass Spectrometry, Plasmid Preparation, Pull Down Assay, Expressing, Immunoprecipitation

    O -GlcNAcylation of Lsp1 controls B-cell apoptosis by enhancing ERK phosphorylation and reducing BCL-2 and BCL-xL expression. ( a ) Flow cytometric analysis of the frequency of Annexin V + Ramos B cells transfected with control siRNA or Lsp1-specific siRNA, and stimulated with anti-IgM (25 μg ml −1 ) for 48 h. Immunoblotting (IB) showing the knockdown of endogenous Lsp1 by Lsp1 siRNA. ( b ) Flow cytometric analysis of the frequency of Annexin V + cells among EGFP + Ramos B cells lentivirally transduced with the indicated vectors and stimulated with anti-IgM (25 μg ml −1 ) with or without the treatment with TG for 48 h. ( c ) Statistical results of the Annexin V + cells described in b are shown. IB showing the equal expression of various variants of Flag-EGFP-tagged Lsp1. ( d ) The frequency of Annexin V + cells in the YFP + gate of mouse splenic B cells transduced with the indicated retroviral vectors and stimulated with anti-IgM (10 μg ml −1 ) for 48 h. ( e ) Statistical results of the Annexin V + cells described in d are shown. IB showing the equal expression of various variants of Flag-tagged Lsp1. The results in c , e represent the mean±s.e.m. ( n =3). * P
    Figure Legend Snippet: O -GlcNAcylation of Lsp1 controls B-cell apoptosis by enhancing ERK phosphorylation and reducing BCL-2 and BCL-xL expression. ( a ) Flow cytometric analysis of the frequency of Annexin V + Ramos B cells transfected with control siRNA or Lsp1-specific siRNA, and stimulated with anti-IgM (25 μg ml −1 ) for 48 h. Immunoblotting (IB) showing the knockdown of endogenous Lsp1 by Lsp1 siRNA. ( b ) Flow cytometric analysis of the frequency of Annexin V + cells among EGFP + Ramos B cells lentivirally transduced with the indicated vectors and stimulated with anti-IgM (25 μg ml −1 ) with or without the treatment with TG for 48 h. ( c ) Statistical results of the Annexin V + cells described in b are shown. IB showing the equal expression of various variants of Flag-EGFP-tagged Lsp1. ( d ) The frequency of Annexin V + cells in the YFP + gate of mouse splenic B cells transduced with the indicated retroviral vectors and stimulated with anti-IgM (10 μg ml −1 ) for 48 h. ( e ) Statistical results of the Annexin V + cells described in d are shown. IB showing the equal expression of various variants of Flag-tagged Lsp1. The results in c , e represent the mean±s.e.m. ( n =3). * P

    Techniques Used: Expressing, Flow Cytometry, Transfection, Transduction

    O -GlcNAcylation of Lsp1 affects the recruitment of PKC-β1. ( a ) Immunoblotting (IB) showing the effects of various doses of a PKC inhibitor, Gö6983 (left panel), or an MK2 inhibitor, MK25 (right panel), on the levels of Lsp1 S243 phosphorylation in anti-IgM (10 μg ml −1 )-stimulated mouse splenic B cells. Inhibitors were added for 1 h, and the cells were collected 24 h after anti-IgM stimulation. ( b ) Statistical results (mean±s.e.m., n =3) showing the mean fluorescent intensity of Annexin V staining from the flow cytometric analysis of splenic B cells expressing the indicated vectors and stimulated with anti-IgM (10 μg ml −1 ) in the presence or absence of Gö6983 at 24 h. ** P
    Figure Legend Snippet: O -GlcNAcylation of Lsp1 affects the recruitment of PKC-β1. ( a ) Immunoblotting (IB) showing the effects of various doses of a PKC inhibitor, Gö6983 (left panel), or an MK2 inhibitor, MK25 (right panel), on the levels of Lsp1 S243 phosphorylation in anti-IgM (10 μg ml −1 )-stimulated mouse splenic B cells. Inhibitors were added for 1 h, and the cells were collected 24 h after anti-IgM stimulation. ( b ) Statistical results (mean±s.e.m., n =3) showing the mean fluorescent intensity of Annexin V staining from the flow cytometric analysis of splenic B cells expressing the indicated vectors and stimulated with anti-IgM (10 μg ml −1 ) in the presence or absence of Gö6983 at 24 h. ** P

    Techniques Used: Staining, Flow Cytometry, Expressing

    6) Product Images from "Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia"

    Article Title: Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia

    Journal: Blood

    doi: 10.1182/blood-2009-03-209247

    Abrogation of Mer expression in the E2A-PBX1 + human B-ALL cell line 697. Cells were infected with lentiviral particles containing short hairpin RNA (shRNA) constructs targeting Mer (shMer1A, shMer1B) or GFP (shControl) as a nonsilencing control. Mer knockdown
    Figure Legend Snippet: Abrogation of Mer expression in the E2A-PBX1 + human B-ALL cell line 697. Cells were infected with lentiviral particles containing short hairpin RNA (shRNA) constructs targeting Mer (shMer1A, shMer1B) or GFP (shControl) as a nonsilencing control. Mer knockdown

    Techniques Used: Expressing, Infection, shRNA, Construct

    7) Product Images from "Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium"

    Article Title: Arginine GlcNAcylation of Rab small GTPases by the pathogen Salmonella Typhimurium

    Journal: Communications Biology

    doi: 10.1038/s42003-020-1005-2

    SseK3 inhibits cytokine secretion and is required for bacterial pathogenesis. a , b Effects of SseK proteins on the secretion of pro-inflammatory cytokines in RAW264.7 cell during S . Typhimurium infection. The cytokine IL-6 ( a ) and TNF ( b ) released during 6–18 h after infection were measured in culture supernatants by ELISA and normalized to CFUs enumerated from each strain at 18 h post infection. Results shown are mean values ± SD (error bar) from three independent experiments. c Effects of SseK proteins or the enzymatic mutant on the secretion of IFN-γ in 293T cells. The content of IFN-γ in culture supernatant and cell lysate was quantified by ELISA and the secretion index was calculated. Results shown are mean values ± SD (error bar) from three independent experiments. d Effects of SseK on the replication of S . Typhimurium in macrophages. RAW264.7 were infected with the indicated S . Typhimurium at a multiplicity of infection of 10. Fold replication was determined by comparing bacterial counts at 2 and 24 h post infection. Results shown are mean values ± SD (error bar) from five independent experiments. e , f Effects of SseK proteins on the bacterial virulence in vivo. Six-week-old C57BL/6 mice were orally infected with the indicated S . Typhimurium strains and killed at 4 days post infection. Bacterial counts in the liver ( e ) and spleen ( f ) were calculated as colony-forming units (CFUs) per gram of tissue. A minimum of five mice was used for each group. Results shown are mean values ± SEM (error bar). n.s., not significant. * P
    Figure Legend Snippet: SseK3 inhibits cytokine secretion and is required for bacterial pathogenesis. a , b Effects of SseK proteins on the secretion of pro-inflammatory cytokines in RAW264.7 cell during S . Typhimurium infection. The cytokine IL-6 ( a ) and TNF ( b ) released during 6–18 h after infection were measured in culture supernatants by ELISA and normalized to CFUs enumerated from each strain at 18 h post infection. Results shown are mean values ± SD (error bar) from three independent experiments. c Effects of SseK proteins or the enzymatic mutant on the secretion of IFN-γ in 293T cells. The content of IFN-γ in culture supernatant and cell lysate was quantified by ELISA and the secretion index was calculated. Results shown are mean values ± SD (error bar) from three independent experiments. d Effects of SseK on the replication of S . Typhimurium in macrophages. RAW264.7 were infected with the indicated S . Typhimurium at a multiplicity of infection of 10. Fold replication was determined by comparing bacterial counts at 2 and 24 h post infection. Results shown are mean values ± SD (error bar) from five independent experiments. e , f Effects of SseK proteins on the bacterial virulence in vivo. Six-week-old C57BL/6 mice were orally infected with the indicated S . Typhimurium strains and killed at 4 days post infection. Bacterial counts in the liver ( e ) and spleen ( f ) were calculated as colony-forming units (CFUs) per gram of tissue. A minimum of five mice was used for each group. Results shown are mean values ± SEM (error bar). n.s., not significant. * P

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Mutagenesis, In Vivo, Mouse Assay

    SseK3 GlcNAcylates Rab small GTPases during S . Typhimurium infection. a Overlap of Arg-GlcNAcylated proteins identified in 293T cells treated with transfected- or Salmonella -delivered SseK3 expression. Protein names in common are listed below. b Scatter plots of protein ratios as a function of their relative abundance. Proteins were immunoprecipitated with anti-Arg-GlcNAc antibody and subjected to LC-MS/MS analysis. The ratio was calculated as spectral counts in S . Typhimurium Δ sseK1/2/3 -pSseK3-infected samples divided by those in Δ sseK1/2/3 -pVec-infected samples. Large ratios indicate preferential detection and modification in HeLa cells infected with SseK3-proficient strains. Red dots correspond to modified Rab GTPase proteins. c Overlap of Arg-GlcNAcylated proteins identified in Salmonella infected HeLa, MEF, and iBMDM cells. Protein names in common are listed below. d Modification of 35 small GTPase proteins by Salmonella -infection-delivered SseK3 was tested and shown in Supplementary Fig. 12 . Shown here is an unrooted phylogenetic tree computed from the amino acid sequences of these tested small GTPase proteins. The analysis was performed by neighbor-joining in MEGA 5.0 software. The scale bar indicates an evolutionary distance of 0.1 aa substitution per position in the sequence. The GlcNAcylated GTPases are shown in red (strong modification) and blue (moderate modification). e Electrospray ionization mass spectrometry (ESI-MS) determination of the total mass of Rab1 immunopurified from Salmonella -infected 293T cells. Δ sseK1/2/3 , SseK1, SseK2, and SseK3 triple-deletion strain of S . Typhimurium SL1344 strain. pSseK3 and pSseK3 DxD, SseK3 and SseK3 DxD (D226A/D228A) rescue plasmid, respectively. Modified Rab1 is shown in red. Asterisk symbols denote the prenylated forms of Rab1. f SseK3 and SseK1 modifiy Rab1 and death domain of TRADD during S . Typhimurium infection, respectively. 293T cells were transfected with Flag-TRADD DD or Flag-Rab1, and then subjected to Salmonella infection. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.
    Figure Legend Snippet: SseK3 GlcNAcylates Rab small GTPases during S . Typhimurium infection. a Overlap of Arg-GlcNAcylated proteins identified in 293T cells treated with transfected- or Salmonella -delivered SseK3 expression. Protein names in common are listed below. b Scatter plots of protein ratios as a function of their relative abundance. Proteins were immunoprecipitated with anti-Arg-GlcNAc antibody and subjected to LC-MS/MS analysis. The ratio was calculated as spectral counts in S . Typhimurium Δ sseK1/2/3 -pSseK3-infected samples divided by those in Δ sseK1/2/3 -pVec-infected samples. Large ratios indicate preferential detection and modification in HeLa cells infected with SseK3-proficient strains. Red dots correspond to modified Rab GTPase proteins. c Overlap of Arg-GlcNAcylated proteins identified in Salmonella infected HeLa, MEF, and iBMDM cells. Protein names in common are listed below. d Modification of 35 small GTPase proteins by Salmonella -infection-delivered SseK3 was tested and shown in Supplementary Fig. 12 . Shown here is an unrooted phylogenetic tree computed from the amino acid sequences of these tested small GTPase proteins. The analysis was performed by neighbor-joining in MEGA 5.0 software. The scale bar indicates an evolutionary distance of 0.1 aa substitution per position in the sequence. The GlcNAcylated GTPases are shown in red (strong modification) and blue (moderate modification). e Electrospray ionization mass spectrometry (ESI-MS) determination of the total mass of Rab1 immunopurified from Salmonella -infected 293T cells. Δ sseK1/2/3 , SseK1, SseK2, and SseK3 triple-deletion strain of S . Typhimurium SL1344 strain. pSseK3 and pSseK3 DxD, SseK3 and SseK3 DxD (D226A/D228A) rescue plasmid, respectively. Modified Rab1 is shown in red. Asterisk symbols denote the prenylated forms of Rab1. f SseK3 and SseK1 modifiy Rab1 and death domain of TRADD during S . Typhimurium infection, respectively. 293T cells were transfected with Flag-TRADD DD or Flag-Rab1, and then subjected to Salmonella infection. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.

    Techniques Used: Infection, Transfection, Expressing, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Modification, Software, Sequencing, Mass Spectrometry, Plasmid Preparation

    Silencing prenylation signals of Rab small GTPases abolishes the GlcNAcylation by SseK3. a Arg-GlcNAcylation detection of Rab1 purified from prokaryotic and eukaryotic cells. Anti-Rab1 is shown as loading control. b , c Effects of GTP loading (Q70L)-, GDP loading (S25N)-, and loss of prenylation (ΔCC and CC-SS) forms of Rab1 on the GlcNAcylation by SseK3 during ectopic expression ( b ) and Salmonella infection ( c ). Lysates were immunoprecipitated with Flag antibody and immunoblotted with indicated antibodies. d Effects of prenylation of Rab1 in a recombinant reaction in vitro. Rab1 with or without prenylation indicates the Rab1 protein purified from 293T cells or E. coli BL21 (DE3) strain, respectively. e Prenylation of some Rab small GTPases is crucial for the GlcNAcylation by SseK3 during Salmonella infection. 293T cells were transfected with Flag-tagged Rab1, Rab8, Rab33, as well as their C-terminal deletion forms, and then subjected to pathogen infection with indicated strains. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP). Representative data from at least three repetitions are shown.
    Figure Legend Snippet: Silencing prenylation signals of Rab small GTPases abolishes the GlcNAcylation by SseK3. a Arg-GlcNAcylation detection of Rab1 purified from prokaryotic and eukaryotic cells. Anti-Rab1 is shown as loading control. b , c Effects of GTP loading (Q70L)-, GDP loading (S25N)-, and loss of prenylation (ΔCC and CC-SS) forms of Rab1 on the GlcNAcylation by SseK3 during ectopic expression ( b ) and Salmonella infection ( c ). Lysates were immunoprecipitated with Flag antibody and immunoblotted with indicated antibodies. d Effects of prenylation of Rab1 in a recombinant reaction in vitro. Rab1 with or without prenylation indicates the Rab1 protein purified from 293T cells or E. coli BL21 (DE3) strain, respectively. e Prenylation of some Rab small GTPases is crucial for the GlcNAcylation by SseK3 during Salmonella infection. 293T cells were transfected with Flag-tagged Rab1, Rab8, Rab33, as well as their C-terminal deletion forms, and then subjected to pathogen infection with indicated strains. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP). Representative data from at least three repetitions are shown.

    Techniques Used: Purification, Expressing, Infection, Immunoprecipitation, Recombinant, In Vitro, Transfection, Western Blot

    8) Product Images from "Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers"

    Article Title: Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09965-w

    Effects of substitutions in Enh-II on the envelope protein expression. Relative ratio of PreS2 and PreS1-mRNA as determined by quantitative real-time PCR ( A ), expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure S2 ( B ); α-Tubulin served as the loading control] ( B ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( C ), following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p
    Figure Legend Snippet: Effects of substitutions in Enh-II on the envelope protein expression. Relative ratio of PreS2 and PreS1-mRNA as determined by quantitative real-time PCR ( A ), expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure S2 ( B ); α-Tubulin served as the loading control] ( B ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( C ), following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection

    Effects of substitutions in reverse transcriptase (rt) and RNaseH (rh) domains of ORF-P on HBV replication. The relative intracellular HBV-DNA level measured by quantitative real-time PCR ( A ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( B ), following transfection of the wt-HBV (pTriEx-Mod/HBV-D-wt) and mutant construct (pTriEx-Mod/HBV-D-mt), having rt and rh substitutions in Huh7 cells. Paired t-test p values; **p
    Figure Legend Snippet: Effects of substitutions in reverse transcriptase (rt) and RNaseH (rh) domains of ORF-P on HBV replication. The relative intracellular HBV-DNA level measured by quantitative real-time PCR ( A ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( B ), following transfection of the wt-HBV (pTriEx-Mod/HBV-D-wt) and mutant construct (pTriEx-Mod/HBV-D-mt), having rt and rh substitutions in Huh7 cells. Paired t-test p values; **p

    Techniques Used: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Construct

    Effects of substitutions in Enh-II on enhancer activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-P-wt-Enh-II and pGL3-P-mt-Enh-II carrying wild-type and mutant Enh-II elements respectively in pGL3-promoter vector. Relative expression of pgRNA ( B ), intracellular HBV-DNA level ( C ) and extracellular HBV-DNA level ( D ), as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p
    Figure Legend Snippet: Effects of substitutions in Enh-II on enhancer activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-P-wt-Enh-II and pGL3-P-mt-Enh-II carrying wild-type and mutant Enh-II elements respectively in pGL3-promoter vector. Relative expression of pgRNA ( B ), intracellular HBV-DNA level ( C ) and extracellular HBV-DNA level ( D ), as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding Enh-II-mutated HBV [HBV/D-mt(Enh-II)] in Huh7 cells. Paired t-test p values; *p

    Techniques Used: Activity Assay, Expressing, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection

    Effects of substitutions in X-promoter on promoter activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-B-HBx-promoter-wt and pGL3-B-HBx-promoter-mt carrying wild-type and mutated X-promoter respectively cloned in pGL3-Basic vector. Relative expression of pgRNA ( B ) and intracellular HBV-DNA level ( C ) as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding X-promoter-mutated HBV [HBV/D-mt(X-prom)] in Huh7 cells. Paired t-test p values; *p
    Figure Legend Snippet: Effects of substitutions in X-promoter on promoter activity, pgRNA expression and HBV-DNA level. ( A ) Relative luciferase activities of the constructs pGL3-B-HBx-promoter-wt and pGL3-B-HBx-promoter-mt carrying wild-type and mutated X-promoter respectively cloned in pGL3-Basic vector. Relative expression of pgRNA ( B ) and intracellular HBV-DNA level ( C ) as measured by quantitative real-time PCR, following transfection of the full-length, wild-type HBV/D (HBV/D-wt) and corresponding X-promoter-mutated HBV [HBV/D-mt(X-prom)] in Huh7 cells. Paired t-test p values; *p

    Techniques Used: Activity Assay, Expressing, Luciferase, Construct, Clone Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection

    Effects of substitutions in ORF-S on HBsAg antigenicity and its detection. ( A ) Antigenicity plot of “a” determinant (aa.124–147) of HBsAg showing decreased antigenicity due to substitutions T125M and P127T as obtained by Kolaskar and Tongaonkar Antigenicity Prediction method. The relative ratio of PreS2/S and PreS1 mRNA determined by quantitative real-time PCR ( B ), the expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure S2 ( A ); α-Tubulin served as the loading control] ( C ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( D ), following transfection of wt-HBV (HBV/D-wt) and mutant construct [HBV/D-mt(HBsAg)], having T125M and P127T substitutions in Huh7 cells. Paired t-test p values; ***p
    Figure Legend Snippet: Effects of substitutions in ORF-S on HBsAg antigenicity and its detection. ( A ) Antigenicity plot of “a” determinant (aa.124–147) of HBsAg showing decreased antigenicity due to substitutions T125M and P127T as obtained by Kolaskar and Tongaonkar Antigenicity Prediction method. The relative ratio of PreS2/S and PreS1 mRNA determined by quantitative real-time PCR ( B ), the expression of HBV envelope proteins by immunoblot assay with mouse monoclonal anti-HBs primary antibody [the cropped gels are shown here for clarity while the full-length blots are presented in Supplementary Figure S2 ( A ); α-Tubulin served as the loading control] ( C ) and HBsAg level [Signal(S)/Cutoff(CO)] in culture supernatant estimated by ELISA ( D ), following transfection of wt-HBV (HBV/D-wt) and mutant construct [HBV/D-mt(HBsAg)], having T125M and P127T substitutions in Huh7 cells. Paired t-test p values; ***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Construct

    Effects of mutations in PreS2 and X-ORFs as well as in Enh-II on generation of ER stress by analyzing GRP78-promoter activity. The full-length, wild-type HBV (HBV/D-wt) along with the mutant variants harbouring mutations in (i) PreS2 and X-ORF [HBV/D-mt(PreS2 + HBX)] and (ii) Enh-II element [HBV/D-mt(Enh-II)] were co-transfected with pGL3-GRP78-promoter construct separately in Huh7 cells and the relative luciferase activities were determined after 48 hrs. Paired t-test p values; *p
    Figure Legend Snippet: Effects of mutations in PreS2 and X-ORFs as well as in Enh-II on generation of ER stress by analyzing GRP78-promoter activity. The full-length, wild-type HBV (HBV/D-wt) along with the mutant variants harbouring mutations in (i) PreS2 and X-ORF [HBV/D-mt(PreS2 + HBX)] and (ii) Enh-II element [HBV/D-mt(Enh-II)] were co-transfected with pGL3-GRP78-promoter construct separately in Huh7 cells and the relative luciferase activities were determined after 48 hrs. Paired t-test p values; *p

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Construct, Luciferase

    9) Product Images from "Characterization of a core fragment of the rhesus monkey TRIM5? protein"

    Article Title: Characterization of a core fragment of the rhesus monkey TRIM5? protein

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-1

    Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).
    Figure Legend Snippet: Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).

    Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot, Molecular Weight

    Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.
    Figure Legend Snippet: Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.

    Techniques Used: Electron Microscopy, Purification, Filtration, Chromatography, Staining, Microscopy, Incubation

    Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.
    Figure Legend Snippet: Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.

    Techniques Used: Generated, Incubation, Molecular Weight

    Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.
    Figure Legend Snippet: Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.

    Techniques Used: Expressing, Solubility, Affinity Column, SDS Page, Staining

    Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.
    Figure Legend Snippet: Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.

    Techniques Used: Size-exclusion Chromatography, Mutagenesis, Purification, Filtration, Flow Cytometry, Staining, Molecular Weight

    Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).
    Figure Legend Snippet: Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).

    Techniques Used: Purification, Expressing, Affinity Chromatography, Lysis, SDS Page, Staining, Affinity Purification, Filtration, Flow Cytometry, Molecular Weight

    10) Product Images from "B cell-intrinsic expression of the HuR RNA-binding protein is required for the T cell-dependent immune response in vivo"

    Article Title: B cell-intrinsic expression of the HuR RNA-binding protein is required for the T cell-dependent immune response in vivo

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500512

    In vitro stimulated HuRΔ/Δ B cells exhibit a mild proliferation defect, enhanced survival, and normal IgH isotype switching. ( A ) Quantification of the fold expansion of splenic B cells from HuRΔ/Δ or HuRf/f mice cultured for 72 hours in LPS and IL-4. Data are from three independent experiments performed on a total of ten animals of each genotype. ( B – E ) Representative flow cytometry analysis and quantification of live cells ( B ), cellular divisions ( C ), cell cycle distribution ( D ), or switched Ig expression ( E ) following culture of HuRΔ/Δ or HuRf/f splenic B cells for 72 hours or where indicated 96 hours in LPS and IL-4. Data are from three or more independent experiments conducted on at least five mice of each genotype.
    Figure Legend Snippet: In vitro stimulated HuRΔ/Δ B cells exhibit a mild proliferation defect, enhanced survival, and normal IgH isotype switching. ( A ) Quantification of the fold expansion of splenic B cells from HuRΔ/Δ or HuRf/f mice cultured for 72 hours in LPS and IL-4. Data are from three independent experiments performed on a total of ten animals of each genotype. ( B – E ) Representative flow cytometry analysis and quantification of live cells ( B ), cellular divisions ( C ), cell cycle distribution ( D ), or switched Ig expression ( E ) following culture of HuRΔ/Δ or HuRf/f splenic B cells for 72 hours or where indicated 96 hours in LPS and IL-4. Data are from three or more independent experiments conducted on at least five mice of each genotype.

    Techniques Used: In Vitro, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

    HuR is required for antibody production and numbers of peritoneal B1 cells but dispensable for in vitro activation in response to anti-IgM and anti-CD40. ( A ) ELISA quantification of IgM or IgG1 secreted by HuRΔ/Δ or HuRf/f splenic B cells during a 72 hour culture in LPS and IL-4. Data are presented as raw values (left graph) or values normalized to the numbers of cells in each culture (right graph). Shown is a representative of three independent experiments. ( B ) Schematic of the final three exons of the μ constant region showing the genomic configuration and the mRNA forms generated by alternative polyadenylation. Arrows above the exons indicate primers used to detect secreted (sec) or membrane-bound (mem) IgM transcripts by qRT-PCR. ( C ) Quantification of IgM transcript variants in LPS and IL-4 stimulated HuRΔ/Δ or HuRf/f splenic B cells presented as their relative abundance to 18S mRNA. Data are from three independent experiments. ( D – E ) Representative flow cytometry analysis and quantification of live cells and cell divisions ( D ) or expression of GC and plasmablast markers ( E ) following culture of HuRf/f or HuRΔ/Δ splenic B cells for 60 hours without stimulation or with stimulation by anti-IgM, anti-CD40, with or without IL-21. Data are representative of two experiments performed on a total of four mice of each genotype. ( F ) ELISA quantification of serum Ig isotypes in non-immunized HuRΔ/Δ and HuRf/f mice. Data are from three or more independent experiments conducted on at least five 6–8 week old mice of each genotype. ( G ) Representative flow cytometry analysis and quantification of peritoneal B1 B cell subsets (CD11b + CD5 + B1a cells and CD11b + CD5 − B1b cells) following gating on live CD19 + lymphocytes. Bar graph shows numbers of total B1 cells from three experiments performed with 6–8 week old mice, 6 HuRf/f and 9 HuRΔ/Δ.
    Figure Legend Snippet: HuR is required for antibody production and numbers of peritoneal B1 cells but dispensable for in vitro activation in response to anti-IgM and anti-CD40. ( A ) ELISA quantification of IgM or IgG1 secreted by HuRΔ/Δ or HuRf/f splenic B cells during a 72 hour culture in LPS and IL-4. Data are presented as raw values (left graph) or values normalized to the numbers of cells in each culture (right graph). Shown is a representative of three independent experiments. ( B ) Schematic of the final three exons of the μ constant region showing the genomic configuration and the mRNA forms generated by alternative polyadenylation. Arrows above the exons indicate primers used to detect secreted (sec) or membrane-bound (mem) IgM transcripts by qRT-PCR. ( C ) Quantification of IgM transcript variants in LPS and IL-4 stimulated HuRΔ/Δ or HuRf/f splenic B cells presented as their relative abundance to 18S mRNA. Data are from three independent experiments. ( D – E ) Representative flow cytometry analysis and quantification of live cells and cell divisions ( D ) or expression of GC and plasmablast markers ( E ) following culture of HuRf/f or HuRΔ/Δ splenic B cells for 60 hours without stimulation or with stimulation by anti-IgM, anti-CD40, with or without IL-21. Data are representative of two experiments performed on a total of four mice of each genotype. ( F ) ELISA quantification of serum Ig isotypes in non-immunized HuRΔ/Δ and HuRf/f mice. Data are from three or more independent experiments conducted on at least five 6–8 week old mice of each genotype. ( G ) Representative flow cytometry analysis and quantification of peritoneal B1 B cell subsets (CD11b + CD5 + B1a cells and CD11b + CD5 − B1b cells) following gating on live CD19 + lymphocytes. Bar graph shows numbers of total B1 cells from three experiments performed with 6–8 week old mice, 6 HuRf/f and 9 HuRΔ/Δ.

    Techniques Used: In Vitro, Activation Assay, Enzyme-linked Immunosorbent Assay, Generated, Size-exclusion Chromatography, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Mouse Assay

    11) Product Images from "Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS"

    Article Title: Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt725

    Activity and selectivity of ASOs in the rodent CNS. ( A ) Allele selective knockdown of muHTT protein with ASO A30 in neuronal cells derived from cortical and striatal tissues of Hu97/18 mouse embryos under free-uptake conditions. ( B–D ) Hu97/18 mice ( n = 4/group) were injected ICV with a single dose of 300 µg of ASO (B and D) or the indicated dose (C) in PBS. Mice were sacrificed after 28 days, the brain was harvested, and a 2 mm coronal slab from each hemisphere (R,L) was analyzed by allelic separation immunoblotting for muHTT and wtHTT protein, and the results were normalized to calnexin protein. (B) Optimized ASOs A25 , A26 , A30 and A31 show similar activity but improved allele selectivity relative to control ASO A1 . (C) Dose response for allele-selective knockdown of muHTT protein following ICV injection of ASO A30 in Hu97/18 mice (D) Immunohistochemical staining for ASO (red) illustrates distribution to al l parts of the brain following a single ICV bolus injection.
    Figure Legend Snippet: Activity and selectivity of ASOs in the rodent CNS. ( A ) Allele selective knockdown of muHTT protein with ASO A30 in neuronal cells derived from cortical and striatal tissues of Hu97/18 mouse embryos under free-uptake conditions. ( B–D ) Hu97/18 mice ( n = 4/group) were injected ICV with a single dose of 300 µg of ASO (B and D) or the indicated dose (C) in PBS. Mice were sacrificed after 28 days, the brain was harvested, and a 2 mm coronal slab from each hemisphere (R,L) was analyzed by allelic separation immunoblotting for muHTT and wtHTT protein, and the results were normalized to calnexin protein. (B) Optimized ASOs A25 , A26 , A30 and A31 show similar activity but improved allele selectivity relative to control ASO A1 . (C) Dose response for allele-selective knockdown of muHTT protein following ICV injection of ASO A30 in Hu97/18 mice (D) Immunohistochemical staining for ASO (red) illustrates distribution to al l parts of the brain following a single ICV bolus injection.

    Techniques Used: Activity Assay, Allele-specific Oligonucleotide, Derivative Assay, Mouse Assay, Injection, Immunohistochemistry, Staining

    12) Product Images from "Protein Kinase A Activity at the Endoplasmic Reticulum Surface Is Responsible for Augmentation of Human ether-a-go-go-related Gene Product (HERG) *"

    Article Title: Protein Kinase A Activity at the Endoplasmic Reticulum Surface Is Responsible for Augmentation of Human ether-a-go-go-related Gene Product (HERG) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.201699

    Targeted PKA inhibitors suppress phosphorylation of phospholamban in rat neonatal cardiomyocytes. Cardiomyocytes infected with p4PKIg (or p4scrg) adenovirus were treated with 50 μ m CPT-cAMP (or DMSO) for 1 h and subjected to Western blot analysis.
    Figure Legend Snippet: Targeted PKA inhibitors suppress phosphorylation of phospholamban in rat neonatal cardiomyocytes. Cardiomyocytes infected with p4PKIg (or p4scrg) adenovirus were treated with 50 μ m CPT-cAMP (or DMSO) for 1 h and subjected to Western blot analysis.

    Techniques Used: Infection, Cycling Probe Technology, Western Blot

    13) Product Images from "Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements *Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements * ♦"

    Article Title: Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements *Cooperative Control of Caspase Recruitment Domain-containing Protein 11 (CARD11) Signaling by an Unusual Array of Redundant Repressive Elements * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.683714

    CARD11 ID contains more than one redundant repressive element. A, schematic of the constructs assayed. B, Jurkat T cells in which CARD11 was stably knocked down ( KD-CARD11 ) or control cells expressing a control shRNA (NT) were transfected with CSK-LacZ and Igκ 2 -IFN-LUC in the presence of expression vectors for the indicated Myc-tagged CARD11 variants and stimulated with anti-CD3/anti-CD28 cross-linking for 5 h as indicated. A two-tailed unpaired Student's t test with unequal variance resulted in p values > 0.05 for the values obtained under unstimulated conditions with the following constructs as compared with that observed with wild-type CARD11: Δ441–488, Δ483–535, Δ530–573, Δ568–616, and Δ649–671. C, HEK293T cells were transfected with the same amounts of each expression vector used in B , and lysates were probed by Western blot ( WB ) using anti-Myc primary antibody to indicate the relative expression level of each variant. β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis. D, KD-CARD11 Jurkat T cell transfections in B were scaled up 9-fold, and each Myc-tagged CARD11 variant was immunoprecipitated ( IP ) by anti-Myc antibody and assayed by Western blot using anti-CARD11 antibody as described under “Experimental Procedures.” β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis.
    Figure Legend Snippet: CARD11 ID contains more than one redundant repressive element. A, schematic of the constructs assayed. B, Jurkat T cells in which CARD11 was stably knocked down ( KD-CARD11 ) or control cells expressing a control shRNA (NT) were transfected with CSK-LacZ and Igκ 2 -IFN-LUC in the presence of expression vectors for the indicated Myc-tagged CARD11 variants and stimulated with anti-CD3/anti-CD28 cross-linking for 5 h as indicated. A two-tailed unpaired Student's t test with unequal variance resulted in p values > 0.05 for the values obtained under unstimulated conditions with the following constructs as compared with that observed with wild-type CARD11: Δ441–488, Δ483–535, Δ530–573, Δ568–616, and Δ649–671. C, HEK293T cells were transfected with the same amounts of each expression vector used in B , and lysates were probed by Western blot ( WB ) using anti-Myc primary antibody to indicate the relative expression level of each variant. β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis. D, KD-CARD11 Jurkat T cell transfections in B were scaled up 9-fold, and each Myc-tagged CARD11 variant was immunoprecipitated ( IP ) by anti-Myc antibody and assayed by Western blot using anti-CARD11 antibody as described under “Experimental Procedures.” β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis.

    Techniques Used: Construct, Stable Transfection, Expressing, shRNA, Transfection, Two Tailed Test, Plasmid Preparation, Western Blot, Variant Assay, Activity Assay, Immunoprecipitation

    14) Product Images from "Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse"

    Article Title: Highly potent anti-CD20-RLI immunocytokine targeting established human B lymphoma in SCID mouse

    Journal: mAbs

    doi: 10.4161/mabs.28699

    Figure 6. CD19 + cells depletion induced by anti-CD20-RLI. Human whole blood from healthy donors was incubated with RLI (large-black-square), RTX (○), RTX + RLI (black-circle) and anti-CD20-RLI (large-white-square) and CD19 + cells depletion was evaluated by flow cytometry. Data are means ± SEM of three experiments.
    Figure Legend Snippet: Figure 6. CD19 + cells depletion induced by anti-CD20-RLI. Human whole blood from healthy donors was incubated with RLI (large-black-square), RTX (○), RTX + RLI (black-circle) and anti-CD20-RLI (large-white-square) and CD19 + cells depletion was evaluated by flow cytometry. Data are means ± SEM of three experiments.

    Techniques Used: Incubation, Flow Cytometry, Cytometry

    Figure 5. CDC and ADCC activities of anti-CD20-RLI. (A) For CDC, CD20 positive Daudi cells were incubated with increasing concentrations of RTX (○), anti-CD20-RLI (black-square) and anti-GD2 (▲) as a negative control, in the presence of human serum as a source of complement. Lysis of Daudi cells was evaluated using 31 Cr release assays. (B) ADCC was evaluated on Raji cells at an E/T ratio of 10:1, in the presence of increasing concentrations of RLI (large-black-square), RTX (○), RTX + RLI (black-circle), anti-GD2 (▲) and anti-CD20-RLI (large-white-square) and using human purified NK cells from healthy donors. Data are means ± SEM of three experiments.
    Figure Legend Snippet: Figure 5. CDC and ADCC activities of anti-CD20-RLI. (A) For CDC, CD20 positive Daudi cells were incubated with increasing concentrations of RTX (○), anti-CD20-RLI (black-square) and anti-GD2 (▲) as a negative control, in the presence of human serum as a source of complement. Lysis of Daudi cells was evaluated using 31 Cr release assays. (B) ADCC was evaluated on Raji cells at an E/T ratio of 10:1, in the presence of increasing concentrations of RLI (large-black-square), RTX (○), RTX + RLI (black-circle), anti-GD2 (▲) and anti-CD20-RLI (large-white-square) and using human purified NK cells from healthy donors. Data are means ± SEM of three experiments.

    Techniques Used: Incubation, Negative Control, Lysis, Purification

    Figure 7. Effect of anti-CD20-RLI on NK-cell activation. CD16A down modulation (A) and CD107 expression (B) induced by RLI, RTX, RTX + RLI and anti-CD20-RLI (10µM) were evaluated in human isolated NK cells after 1h (○), 2h (▲) or 3h (large-black-square) incubation time. A combination of PMA and Calcium Ionophore was used as positive control. Data are means ± SEM of three experiments.
    Figure Legend Snippet: Figure 7. Effect of anti-CD20-RLI on NK-cell activation. CD16A down modulation (A) and CD107 expression (B) induced by RLI, RTX, RTX + RLI and anti-CD20-RLI (10µM) were evaluated in human isolated NK cells after 1h (○), 2h (▲) or 3h (large-black-square) incubation time. A combination of PMA and Calcium Ionophore was used as positive control. Data are means ± SEM of three experiments.

    Techniques Used: Activation Assay, Expressing, Isolation, Incubation, Positive Control

    Figure 2. Production and purification of the anti-CD20-RLI ICK. (A) (B) SDS-PAGE under non reducing (left panel) and reducing (middle panel) conditions (Lane 1: 5µg of RTX, line 2: 5µg of anti-CD20-RLI); western blot analysis using anti-IgG Ab (right panel, line 1: 0.1µg of anti-CD20-RLI) or anti-IL-15 Ab (right panel, line 2: 0.1µg of anti-CD20-RLI). kDa: kilo Dalton; Mw: Molecular weight. (C) gel-filtration analysis of RTX (left panel) or affinity-purified anti-CD20-RLI (right panel) revealing mainly monomer.
    Figure Legend Snippet: Figure 2. Production and purification of the anti-CD20-RLI ICK. (A) (B) SDS-PAGE under non reducing (left panel) and reducing (middle panel) conditions (Lane 1: 5µg of RTX, line 2: 5µg of anti-CD20-RLI); western blot analysis using anti-IgG Ab (right panel, line 1: 0.1µg of anti-CD20-RLI) or anti-IL-15 Ab (right panel, line 2: 0.1µg of anti-CD20-RLI). kDa: kilo Dalton; Mw: Molecular weight. (C) gel-filtration analysis of RTX (left panel) or affinity-purified anti-CD20-RLI (right panel) revealing mainly monomer.

    Techniques Used: Purification, SDS Page, Western Blot, Molecular Weight, Filtration, Affinity Purification

    Figure 9. Effect of anti-CD20-RLI on survival of tumor-bearing SCID mice. Mice were inoculated intravenously with Raji cells (2.5x10 6 ). At days 5, 10, 15 and 20 after tumor inoculation, groups of 10 mice were treated with (A) saline (open triangle), low dose (12µg, open circle) or conventional therapeutic dose (200µg, filled gray circle) of RTX or (B) with equimolar dose of saline, RLI (2µg), RTX (12µg), RTX + RLI, anti-CD20-RLI (16µg) or anti-GD2-RLI (16µg). Mice were monitored daily and sacrificed at the onset of hind leg paralysis. Percent survival of mice after treatment with saline (▽), RLI (large-black-square), RTX (○), RTX + RLI (black-circle), anti-CD20-RLI (large-white-square) or anti-GD2-RLI (◇). *, P
    Figure Legend Snippet: Figure 9. Effect of anti-CD20-RLI on survival of tumor-bearing SCID mice. Mice were inoculated intravenously with Raji cells (2.5x10 6 ). At days 5, 10, 15 and 20 after tumor inoculation, groups of 10 mice were treated with (A) saline (open triangle), low dose (12µg, open circle) or conventional therapeutic dose (200µg, filled gray circle) of RTX or (B) with equimolar dose of saline, RLI (2µg), RTX (12µg), RTX + RLI, anti-CD20-RLI (16µg) or anti-GD2-RLI (16µg). Mice were monitored daily and sacrificed at the onset of hind leg paralysis. Percent survival of mice after treatment with saline (▽), RLI (large-black-square), RTX (○), RTX + RLI (black-circle), anti-CD20-RLI (large-white-square) or anti-GD2-RLI (◇). *, P

    Techniques Used: Mouse Assay

    Figure 3. Characterization of the anti-CD20-RLI ICK. (A) Specific binding of anti-CD20-RLI and RTX antibodies revealed by flow cytometry: Raji (left panel) , Kit225 (middle panel) and 32Dβ (right panel) cells. Control Ab (filled pink); RTX (green line), anti-CD20-RLI (orange line) and anti-GD2-RLI (blue line). (B) Binding of RTX and anti-CD20-RLI to human CD16-transduced NK-92 cells. CD16-transduced NK-92 cells were incubated with varying concentrations of RTX (○), RLI (■), anti-CD20-RLI (black-square) or the association of RTX and RLI (●) for 30 min at 4 °C followed by FITC-conjugated anti-CD16 3G8 mAb and then analyzed by flow cytometry. Percentages of inhibition of 3G8 binding were calculated as described in “Methods.” (C) Binding affinities of RLI and anti-CD20-RLI for IL-15Rβ/γ. SPR sensorgrams of binding to immobilized soluble IL-15Rβ/γ with increasing concentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200nM) of RLI (left panel) or anti-CD20-RLI (right panel).
    Figure Legend Snippet: Figure 3. Characterization of the anti-CD20-RLI ICK. (A) Specific binding of anti-CD20-RLI and RTX antibodies revealed by flow cytometry: Raji (left panel) , Kit225 (middle panel) and 32Dβ (right panel) cells. Control Ab (filled pink); RTX (green line), anti-CD20-RLI (orange line) and anti-GD2-RLI (blue line). (B) Binding of RTX and anti-CD20-RLI to human CD16-transduced NK-92 cells. CD16-transduced NK-92 cells were incubated with varying concentrations of RTX (○), RLI (■), anti-CD20-RLI (black-square) or the association of RTX and RLI (●) for 30 min at 4 °C followed by FITC-conjugated anti-CD16 3G8 mAb and then analyzed by flow cytometry. Percentages of inhibition of 3G8 binding were calculated as described in “Methods.” (C) Binding affinities of RLI and anti-CD20-RLI for IL-15Rβ/γ. SPR sensorgrams of binding to immobilized soluble IL-15Rβ/γ with increasing concentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200nM) of RLI (left panel) or anti-CD20-RLI (right panel).

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Incubation, Inhibition, SPR Assay

    15) Product Images from "Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable *"

    Article Title: Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.615310

    Mitosis-specific phosphorylation of Ser-1126 decreases the solubility of separase. A , aggravated aggregation tendency of separase ( Sep. ) in mitosis. Synchronized interphase ( Int ., Interph. ) or prometaphase ( Mit .) transgenic HEK293 cells induced to express
    Figure Legend Snippet: Mitosis-specific phosphorylation of Ser-1126 decreases the solubility of separase. A , aggravated aggregation tendency of separase ( Sep. ) in mitosis. Synchronized interphase ( Int ., Interph. ) or prometaphase ( Mit .) transgenic HEK293 cells induced to express

    Techniques Used: Solubility, Transgenic Assay

    Positive and negative effects of Cdk1-cyclin B1 define the minimal requirements of separase regulation in mitosis. A , WT separase and mutants S1126A and ΔCLD exhibit some, high, or no cohesin cleavage activity upon isolation from prometaphase-arrested
    Figure Legend Snippet: Positive and negative effects of Cdk1-cyclin B1 define the minimal requirements of separase regulation in mitosis. A , WT separase and mutants S1126A and ΔCLD exhibit some, high, or no cohesin cleavage activity upon isolation from prometaphase-arrested

    Techniques Used: Activity Assay, Isolation

    16) Product Images from "Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo"

    Article Title: Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.10.002

    Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). .
    Figure Legend Snippet: Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). .

    Techniques Used: Binding Assay, Transfection, Staining, Expressing, Western Blot, Migration, Whisker Assay, Construct, Mutagenesis, MANN-WHITNEY

    17) Product Images from "Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10 *"

    Article Title: Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.560938

    sgp130-E10 is conserved in many mammals and most abundantly expressed in blood cells. A , schematic overview of gp130 and the novel sgp130-E10 variant in comparison with the three transcripts formed by alternative polyadenylation. Exons are indicated by
    Figure Legend Snippet: sgp130-E10 is conserved in many mammals and most abundantly expressed in blood cells. A , schematic overview of gp130 and the novel sgp130-E10 variant in comparison with the three transcripts formed by alternative polyadenylation. Exons are indicated by

    Techniques Used: Variant Assay

    Analysis of the binding of Hyper-IL-6 to captured sgp130Fc and gp130-E10Fc by surface plasmon resonance spectroscopy. A , sgp130-E10Fc and B , sgp130Fc were captured to immobilized human Fc antibody, and Hyper-IL-6 was injected for 180 s at concentrations
    Figure Legend Snippet: Analysis of the binding of Hyper-IL-6 to captured sgp130Fc and gp130-E10Fc by surface plasmon resonance spectroscopy. A , sgp130-E10Fc and B , sgp130Fc were captured to immobilized human Fc antibody, and Hyper-IL-6 was injected for 180 s at concentrations

    Techniques Used: Binding Assay, SPR Assay, Spectroscopy, Injection

    Specific detection of sgp130-E10 by the monoclonal sgp130-E10 antibody E10/1 in PBMCs and serum. A , Western blot ( WB ) analysis of sgp130-E10Fc and sgp130Fc in different concentrations. The monoclonal sgp130-E10 antibody E10/1 specifically detected recombinant
    Figure Legend Snippet: Specific detection of sgp130-E10 by the monoclonal sgp130-E10 antibody E10/1 in PBMCs and serum. A , Western blot ( WB ) analysis of sgp130-E10Fc and sgp130Fc in different concentrations. The monoclonal sgp130-E10 antibody E10/1 specifically detected recombinant

    Techniques Used: Western Blot, Recombinant

    sgp130-E10Fc interacts with Hyper-IL-6, but not with IL-6 alone. A , Coomassie Blue stains of sgp130-E10Fc and sgp130Fc. Different amounts from 1 to 20 μg of sgp130-E10Fc and sgp130Fc were separated by SDS-PAGE and visualized by Coomassie Blue
    Figure Legend Snippet: sgp130-E10Fc interacts with Hyper-IL-6, but not with IL-6 alone. A , Coomassie Blue stains of sgp130-E10Fc and sgp130Fc. Different amounts from 1 to 20 μg of sgp130-E10Fc and sgp130Fc were separated by SDS-PAGE and visualized by Coomassie Blue

    Techniques Used: SDS Page

    Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His
    Figure Legend Snippet: Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His

    Techniques Used: Immunoprecipitation, Transfection

    Purified sgp130-E10Fc is biologically active, but inferior to sgp130Fc in terms of binding to Hyper-IL-6, activity and stability. A , competitive ELISA with coated sgp130Fc and competition with either sgp130Fc or sgp130-E10Fc for Hyper-IL-6 binding. Standard
    Figure Legend Snippet: Purified sgp130-E10Fc is biologically active, but inferior to sgp130Fc in terms of binding to Hyper-IL-6, activity and stability. A , competitive ELISA with coated sgp130Fc and competition with either sgp130Fc or sgp130-E10Fc for Hyper-IL-6 binding. Standard

    Techniques Used: Purification, Binding Assay, Activity Assay, Competitive ELISA

    sgp130-E10 protein Binds to IL-6/Soluble IL-6R (Hyper-IL-6), but Not to IL-6 Alone
    Figure Legend Snippet: sgp130-E10 protein Binds to IL-6/Soluble IL-6R (Hyper-IL-6), but Not to IL-6 Alone

    Techniques Used:

    18) Product Images from "EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice"

    Article Title: EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice

    Journal: Endocrine-related cancer

    doi: 10.1530/ERC-16-0142

    Insulin Receptor Knockdown inhibited pulmonary metastases in hyperinsulinemic mice. Lungs were fixed, paraffin embedded and sectioned. Representative images of H E stained lung sections from Rag/WT and Rag/MKR mice injected with LCC6 Ctrl and
    Figure Legend Snippet: Insulin Receptor Knockdown inhibited pulmonary metastases in hyperinsulinemic mice. Lungs were fixed, paraffin embedded and sectioned. Representative images of H E stained lung sections from Rag/WT and Rag/MKR mice injected with LCC6 Ctrl and

    Techniques Used: Mouse Assay, Staining, Injection

    Silencing the insulin receptor leads to decreased tumor growth. 8–10 week old Rag/WT control and Rag/MKR hyperinsulinemic mice were injected with either 5×10 6 LCC6 control (Ctrl) or 5×10 6 LCC6 insulin receptor knockdown (IRKD)
    Figure Legend Snippet: Silencing the insulin receptor leads to decreased tumor growth. 8–10 week old Rag/WT control and Rag/MKR hyperinsulinemic mice were injected with either 5×10 6 LCC6 control (Ctrl) or 5×10 6 LCC6 insulin receptor knockdown (IRKD)

    Techniques Used: Mouse Assay, Injection

    Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p
    Figure Legend Snippet: Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p

    Techniques Used: Expressing, Mouse Assay

    Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry
    Figure Legend Snippet: Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry

    Techniques Used: Western Blot, Expressing

    Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading
    Figure Legend Snippet: Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading

    Techniques Used: Western Blot, Expressing

    19) Product Images from "Dual Interaction of JAM-C with JAM-B and ?M?2 Integrin: Function in Junctional Complexes and Leukocyte Adhesion D⃞"

    Article Title: Dual Interaction of JAM-C with JAM-B and ?M?2 Integrin: Function in Junctional Complexes and Leukocyte Adhesion D⃞

    Journal:

    doi: 10.1091/mbc.E05-04-0310

    Recruitment of JAM-C at cell-cell contacts depletes the apical pool of the protein. (A) CHO cells transfected with EGFP JAM-C (green) were mixed with JAM-C full-length (red) or (B) JAM-B-expressing cells (red) and stained with polyclonal antibodies against
    Figure Legend Snippet: Recruitment of JAM-C at cell-cell contacts depletes the apical pool of the protein. (A) CHO cells transfected with EGFP JAM-C (green) were mixed with JAM-C full-length (red) or (B) JAM-B-expressing cells (red) and stained with polyclonal antibodies against

    Techniques Used: Transfection, Expressing, Staining

    Anti-JAM-C mAb blocks JAM-B/JAM-C interaction and modulates JAM-C localization. (A) Cell lysates obtained from JAM-B-EGFP-transfected MDCK cells were precipitated with beads coupled to soluble JAM-C in the presence of irrelevant anti-PECAM antibody (GC51)
    Figure Legend Snippet: Anti-JAM-C mAb blocks JAM-B/JAM-C interaction and modulates JAM-C localization. (A) Cell lysates obtained from JAM-B-EGFP-transfected MDCK cells were precipitated with beads coupled to soluble JAM-C in the presence of irrelevant anti-PECAM antibody (GC51)

    Techniques Used: Transfection

    Dynamic of JAM-C junctional recruitment by JAM-B in CHO-transfected cells. (A) EGFP JAM-C CHO cells were mixed with JAM-B CHO cells at a ratio 1:1. FRAP experiments of EGFP JAM-C engaged in homophilic interaction (top panel) or heterophilic interaction
    Figure Legend Snippet: Dynamic of JAM-C junctional recruitment by JAM-B in CHO-transfected cells. (A) EGFP JAM-C CHO cells were mixed with JAM-B CHO cells at a ratio 1:1. FRAP experiments of EGFP JAM-C engaged in homophilic interaction (top panel) or heterophilic interaction

    Techniques Used: Transfection

    JAM-C interacts heterophilically with JAM-B through its V domain, and monomeric JAM-B dissociates JAM-C homodimers to form heterodimers. (A) JAM-B-EGFP-transfected MDCK cells were mixed with nontransfected cells and stained with soluble JAM-C consisting
    Figure Legend Snippet: JAM-C interacts heterophilically with JAM-B through its V domain, and monomeric JAM-B dissociates JAM-C homodimers to form heterodimers. (A) JAM-B-EGFP-transfected MDCK cells were mixed with nontransfected cells and stained with soluble JAM-C consisting

    Techniques Used: Transfection, Staining

    JAM-C is recruited at cell-cell contacts by JAM-B. FLAG-JAM-B- and JAM-C-EGFP-transfected MDCK cells were mixed and the localization of the molecules was visualized by immunofluorescent labeling. (A) Negative control for JAM-B staining with polyclonal
    Figure Legend Snippet: JAM-C is recruited at cell-cell contacts by JAM-B. FLAG-JAM-B- and JAM-C-EGFP-transfected MDCK cells were mixed and the localization of the molecules was visualized by immunofluorescent labeling. (A) Negative control for JAM-B staining with polyclonal

    Techniques Used: Transfection, Labeling, Negative Control, Staining

    20) Product Images from "The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models"

    Article Title: The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2017.06.001

    ADCC, but not CDC, is augmented by the combination of IMGN529 and rituximab. ADCC assays were performed by incubating target U-2932 (A) or SU-DHL-4 (B) cells with human NK effector cells at E/T ratio of 3:1 or 4:1 and measuring LDH release. Cells were exposed to increasing concentrations of IMGN529 in the presence or absence of varying concentrations of rituximab or control IgG, as indicated. Percent specific lysis was calculated, and data shown are the mean of three experiments. (C) CDC activity against U-2932 cells ( left panel ) and SU-DHL-4 cells ( right panel ) was determined following incubating cells for 2 hours with increasing concentrations of rituximab +/− 5 μg/mL of IMGN529 or IgG1-SMCC-DM1 in the presence of human complement. Cell viability was assessed by alamarBlue assay.
    Figure Legend Snippet: ADCC, but not CDC, is augmented by the combination of IMGN529 and rituximab. ADCC assays were performed by incubating target U-2932 (A) or SU-DHL-4 (B) cells with human NK effector cells at E/T ratio of 3:1 or 4:1 and measuring LDH release. Cells were exposed to increasing concentrations of IMGN529 in the presence or absence of varying concentrations of rituximab or control IgG, as indicated. Percent specific lysis was calculated, and data shown are the mean of three experiments. (C) CDC activity against U-2932 cells ( left panel ) and SU-DHL-4 cells ( right panel ) was determined following incubating cells for 2 hours with increasing concentrations of rituximab +/− 5 μg/mL of IMGN529 or IgG1-SMCC-DM1 in the presence of human complement. Cell viability was assessed by alamarBlue assay.

    Techniques Used: Lysis, Activity Assay, Alamar Blue Assay

    21) Product Images from "Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT‐independent manner"

    Article Title: Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT‐independent manner

    Journal: Cellular Microbiology

    doi: 10.1111/j.1462-5822.2010.01557.x

    HBV budding requires ESCRT, but not Alix. A. DN Alix blocks HBV budding. HuH‐7 cells were co‐transfected with the HBV replicon and empty plasmid DNA (Control), HA‐tagged WT Alix or the GFP‐tagged Alix mutant (Alix.GFP) at a 1:3 DNA weight ratio respectively. Three days post transfection, cellular supernatants (Medium; black columns) and cytoplasmic extracts prepared with Triton X‐100 (Cell; grey columns) were harvested. HBV release was detected by envelope‐specific immunoprecipitation of supernatants and real‐time PCR of the viral genomes. Non‐enveloped cytoplasmic nucleocapsids were immunoprecipitated with anti‐capsid antibodies (K45) and analysed by PCR. PCR results were demonstrated in per cent amount relative to control‐transfected cells. B. DN Alix blocks HIV.Gag budding. GFP‐tagged HIV.Gag was co‐transfected with control DNA or the Alix.GFP construct at a 1:3 ratio. NP‐40 lysates and VLPs harvested from the supernatants were analysed by GFP‐ and β‐actin‐specific immunoblotting. C. Alix depletion does not inhibit HBV budding. HuH‐7 cells were transfected with control siRNA or siRNA against Alix. Two days later, cells were retransfected with the HBV replicon and harvested after additional 3 days. Intracellular nucleocapsids and extracellular virions were assayed as in (A). Mean results of four PCR reactions are demonstrated in per cent amount relative to control‐treated cells (left). To probe for Alix depletion, the same lysates were immunoblotted with anti‐Alix and anti‐β‐actin antibodies (right).
    Figure Legend Snippet: HBV budding requires ESCRT, but not Alix. A. DN Alix blocks HBV budding. HuH‐7 cells were co‐transfected with the HBV replicon and empty plasmid DNA (Control), HA‐tagged WT Alix or the GFP‐tagged Alix mutant (Alix.GFP) at a 1:3 DNA weight ratio respectively. Three days post transfection, cellular supernatants (Medium; black columns) and cytoplasmic extracts prepared with Triton X‐100 (Cell; grey columns) were harvested. HBV release was detected by envelope‐specific immunoprecipitation of supernatants and real‐time PCR of the viral genomes. Non‐enveloped cytoplasmic nucleocapsids were immunoprecipitated with anti‐capsid antibodies (K45) and analysed by PCR. PCR results were demonstrated in per cent amount relative to control‐transfected cells. B. DN Alix blocks HIV.Gag budding. GFP‐tagged HIV.Gag was co‐transfected with control DNA or the Alix.GFP construct at a 1:3 ratio. NP‐40 lysates and VLPs harvested from the supernatants were analysed by GFP‐ and β‐actin‐specific immunoblotting. C. Alix depletion does not inhibit HBV budding. HuH‐7 cells were transfected with control siRNA or siRNA against Alix. Two days later, cells were retransfected with the HBV replicon and harvested after additional 3 days. Intracellular nucleocapsids and extracellular virions were assayed as in (A). Mean results of four PCR reactions are demonstrated in per cent amount relative to control‐treated cells (left). To probe for Alix depletion, the same lysates were immunoblotted with anti‐Alix and anti‐β‐actin antibodies (right).

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Construct

    Excess Alix enhances HBV naked (nucleo)capsid release. A. Core (lanes 1 and 2) or DPAF‐tagged core (lanes 3 and 4) was co‐transfected with control DNA or HA‐tagged Alix at a 1:3 DNA ratio respectively. Cell extracts were prepared with SDS and analysed by HA‐specific immunoblotting to monitor expression of Alix. For detection of core, the core antiserum (K46) (lanes 1 and 2) or the DPAF antibody (lanes 3 and 4) was used. In either case, non‐specifically stained bands served as a control for gel loading (Load). Capsids harvested from the culture media were analysed in the same manner. The experiments were repeated three times, and capsids released into the supernatants were quantified and demonstrated in per cent amount relative to control cells. To probe for cell lysis, supernatants were assayed for LDH activity. B. The pHBV replicon was co‐transfected with Alix or control plasmid DNA. Cell supernatants were immunoprecipitated with the capsid‐specific antiserum (K45) prior to PCR measurement of the viral DNA. Mean PCR results are demonstrated in per cent amount relative to control‐transfected cells. C. The core mutants Core.K96A (lanes 1 and 2) and CoreΔPPAY (lanes 3 and 4) were subjected to the co‐transfection assay exactly as in (A). Cell lysates and media concentrated by either ultracentrifugation (UC) or TCA precipitation (TCA) were immunoblotted with the core antiserum (K46). D. An HA‐tagged version of the HBV small envelope protein (S.HA) was transfected into HuH‐7 cells together with Alix or a control construct at a 1:3 ratio respectively. Amounts of S were examined by ELISA and are expressed as mean units of optical density at 492 nm ( n = 3).
    Figure Legend Snippet: Excess Alix enhances HBV naked (nucleo)capsid release. A. Core (lanes 1 and 2) or DPAF‐tagged core (lanes 3 and 4) was co‐transfected with control DNA or HA‐tagged Alix at a 1:3 DNA ratio respectively. Cell extracts were prepared with SDS and analysed by HA‐specific immunoblotting to monitor expression of Alix. For detection of core, the core antiserum (K46) (lanes 1 and 2) or the DPAF antibody (lanes 3 and 4) was used. In either case, non‐specifically stained bands served as a control for gel loading (Load). Capsids harvested from the culture media were analysed in the same manner. The experiments were repeated three times, and capsids released into the supernatants were quantified and demonstrated in per cent amount relative to control cells. To probe for cell lysis, supernatants were assayed for LDH activity. B. The pHBV replicon was co‐transfected with Alix or control plasmid DNA. Cell supernatants were immunoprecipitated with the capsid‐specific antiserum (K45) prior to PCR measurement of the viral DNA. Mean PCR results are demonstrated in per cent amount relative to control‐transfected cells. C. The core mutants Core.K96A (lanes 1 and 2) and CoreΔPPAY (lanes 3 and 4) were subjected to the co‐transfection assay exactly as in (A). Cell lysates and media concentrated by either ultracentrifugation (UC) or TCA precipitation (TCA) were immunoblotted with the core antiserum (K46). D. An HA‐tagged version of the HBV small envelope protein (S.HA) was transfected into HuH‐7 cells together with Alix or a control construct at a 1:3 ratio respectively. Amounts of S were examined by ELISA and are expressed as mean units of optical density at 492 nm ( n = 3).

    Techniques Used: Transfection, Expressing, Staining, Lysis, Activity Assay, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction, Cotransfection, TCA Precipitation, Construct, Enzyme-linked Immunosorbent Assay

    22) Product Images from "IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells"

    Article Title: IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01141-13

    IscR-regulated genes possibly involved in the pathogenesis of V. vulnificus . Twelve genes possibly involved in the pathogenesis of V. vulnificus were chosen from the pool of the IscR regulon members predicted by microarray analysis. Regulation of their
    Figure Legend Snippet: IscR-regulated genes possibly involved in the pathogenesis of V. vulnificus . Twelve genes possibly involved in the pathogenesis of V. vulnificus were chosen from the pool of the IscR regulon members predicted by microarray analysis. Regulation of their

    Techniques Used: Microarray

    Induction of iscR expression by INT-407 host cells. Wild-type V. vulnificus was exposed to various numbers of INT-407 cells for 30 min as indicated and then used to isolate total RNAs and proteins as described in Materials and Methods. (A) The iscR mRNA
    Figure Legend Snippet: Induction of iscR expression by INT-407 host cells. Wild-type V. vulnificus was exposed to various numbers of INT-407 cells for 30 min as indicated and then used to isolate total RNAs and proteins as described in Materials and Methods. (A) The iscR mRNA

    Techniques Used: Expressing

    Adhesion of the V. vulnificus strains. (A) INT-407 cells were infected at an MOI of 10 with the V. vulnificus strains as indicated. After 30 min, adherent bacteria were enumerated, and results are presented as the numbers of bacteria per well of the tissue
    Figure Legend Snippet: Adhesion of the V. vulnificus strains. (A) INT-407 cells were infected at an MOI of 10 with the V. vulnificus strains as indicated. After 30 min, adherent bacteria were enumerated, and results are presented as the numbers of bacteria per well of the tissue

    Techniques Used: Infection

    Effects of scavenging ROS on host cell induction of iscR expression. (A and B) Wild-type V. vulnificus grown to an A 600 of 0.5 was exposed to MEMF (control), 4 × 10 6 INT-407 cells, or 4 × 10 6 INT-407 cells preincubated with NAC for 30
    Figure Legend Snippet: Effects of scavenging ROS on host cell induction of iscR expression. (A and B) Wild-type V. vulnificus grown to an A 600 of 0.5 was exposed to MEMF (control), 4 × 10 6 INT-407 cells, or 4 × 10 6 INT-407 cells preincubated with NAC for 30

    Techniques Used: Expressing

    Motility of the V. vulnificus strains. (A) The areas of motility of the strains grown at 30°C for 24 h on plates with LBS and 0.3% agar were photographed. (B) The diameters of motility areas are the means plus SEM of results from three independent
    Figure Legend Snippet: Motility of the V. vulnificus strains. (A) The areas of motility of the strains grown at 30°C for 24 h on plates with LBS and 0.3% agar were photographed. (B) The diameters of motility areas are the means plus SEM of results from three independent

    Techniques Used:

    Cytotoxicity and mouse mortality of V. vulnificus . (A) INT-407 cells were infected with the V. vulnificus strains at an MOI of 10. The cytotoxicity was determined by an LDH release assay and expressed using the total LDH released from the cells completely
    Figure Legend Snippet: Cytotoxicity and mouse mortality of V. vulnificus . (A) INT-407 cells were infected with the V. vulnificus strains at an MOI of 10. The cytotoxicity was determined by an LDH release assay and expressed using the total LDH released from the cells completely

    Techniques Used: Infection, Lactate Dehydrogenase Assay

    Growth of V. vulnificus under oxidative stress. The V. vulnificus strains were compared for their ability to grow on LBS plates supplemented without oxidants (LBS) or with 250 μM H 2 O 2 or 60 μM t -BOOH. Serial 10-fold dilutions of each culture
    Figure Legend Snippet: Growth of V. vulnificus under oxidative stress. The V. vulnificus strains were compared for their ability to grow on LBS plates supplemented without oxidants (LBS) or with 250 μM H 2 O 2 or 60 μM t -BOOH. Serial 10-fold dilutions of each culture

    Techniques Used:

    Hemolytic activities of V. vulnificus . An aliquot of the culture supernatants of V. vulnificus strains was mixed with an equal volume of hRBCs and then incubated at 37°C for 20 min. The lysis of hRBCs was determined, and results are presented
    Figure Legend Snippet: Hemolytic activities of V. vulnificus . An aliquot of the culture supernatants of V. vulnificus strains was mixed with an equal volume of hRBCs and then incubated at 37°C for 20 min. The lysis of hRBCs was determined, and results are presented

    Techniques Used: Incubation, Lysis

    Induction of iscR expression by H 2 O 2 . Total RNAs and proteins were isolated from wild-type V. vulnificus , grown anaerobically to an A 600 of 0.5, and then exposed to various levels of H 2 O 2 for 10 min as indicated. (A) The iscR mRNA levels were determined
    Figure Legend Snippet: Induction of iscR expression by H 2 O 2 . Total RNAs and proteins were isolated from wild-type V. vulnificus , grown anaerobically to an A 600 of 0.5, and then exposed to various levels of H 2 O 2 for 10 min as indicated. (A) The iscR mRNA levels were determined

    Techniques Used: Expressing, Isolation

    23) Product Images from "Niacin analogue, 6-Aminonicotinamide, a novel inhibitor of hepatitis B virus replication and HBsAg production"

    Article Title: Niacin analogue, 6-Aminonicotinamide, a novel inhibitor of hepatitis B virus replication and HBsAg production

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.10.022

    Effect of 6-AN on the promoter activity of HBV genes. (a-b) HBV-infected PHH cells were treated with 7-aminoactinomycin D (5 μ g/ml). Total HBV RNAs and 3.5-kb RNA were quantified by RT-qPCR and the amount of RNA at time zero was set at 100%. (c-d) HBV-infected PHH cells incubated with 0.2 mM EU for another 24 h after 6-AN treated 6 days. The newly synthesized EU-labeled RNA was purified from the total RNA, and EU-labeled HBV RNAs were quantified by RT-qPCR. (e) HepAD38 cells were cultured with Tet (2 μ g/ml) and ETV (25 nM) during 12 days to eliminate rcDNA, the source of cccDNA. And then, 6-AN was added into the medium, changed every two days. After 6-AN treated 4 days and 6 days, cells were collected to detect the level of cccDNA by Taq man probe real-time PCR. (f) The activities of four HBV promotors were detected by dual-luciferase reporter assay system after 6-AN treated 36 h in Huh-7 cells. (g)Twenty transcription factors related to HBV SpI, SpII and core promotors were screened by RT-qPCR in HepG2-NTCP cells after treated 6-AN 6 days, PPARα were screened out from it. (h) Western blot assay further proved 6-AN reduced the expression of PPARα. (i) Western blotting of PPARα proteins in cells transfected with pcDNA3.1 vector expressing PPARα. GAPDH was used as a loading control. (j-l) The activities of HBV promoters (j) and the level of HBV RNAs (i-m) were detected in Huh-7 cells and HBV infected HepG2-NTCP cells after overexpressed PPARα or 6-AN treated. PPARα overexpression rescued the inhibiting effect caused by 6-AN treated. Results are expressed as the average of three independent experiments ( n = 3 per group). The mean value ± standard error is indicated. (* P
    Figure Legend Snippet: Effect of 6-AN on the promoter activity of HBV genes. (a-b) HBV-infected PHH cells were treated with 7-aminoactinomycin D (5 μ g/ml). Total HBV RNAs and 3.5-kb RNA were quantified by RT-qPCR and the amount of RNA at time zero was set at 100%. (c-d) HBV-infected PHH cells incubated with 0.2 mM EU for another 24 h after 6-AN treated 6 days. The newly synthesized EU-labeled RNA was purified from the total RNA, and EU-labeled HBV RNAs were quantified by RT-qPCR. (e) HepAD38 cells were cultured with Tet (2 μ g/ml) and ETV (25 nM) during 12 days to eliminate rcDNA, the source of cccDNA. And then, 6-AN was added into the medium, changed every two days. After 6-AN treated 4 days and 6 days, cells were collected to detect the level of cccDNA by Taq man probe real-time PCR. (f) The activities of four HBV promotors were detected by dual-luciferase reporter assay system after 6-AN treated 36 h in Huh-7 cells. (g)Twenty transcription factors related to HBV SpI, SpII and core promotors were screened by RT-qPCR in HepG2-NTCP cells after treated 6-AN 6 days, PPARα were screened out from it. (h) Western blot assay further proved 6-AN reduced the expression of PPARα. (i) Western blotting of PPARα proteins in cells transfected with pcDNA3.1 vector expressing PPARα. GAPDH was used as a loading control. (j-l) The activities of HBV promoters (j) and the level of HBV RNAs (i-m) were detected in Huh-7 cells and HBV infected HepG2-NTCP cells after overexpressed PPARα or 6-AN treated. PPARα overexpression rescued the inhibiting effect caused by 6-AN treated. Results are expressed as the average of three independent experiments ( n = 3 per group). The mean value ± standard error is indicated. (* P

    Techniques Used: Activity Assay, Infection, Quantitative RT-PCR, Incubation, Synthesized, Labeling, Purification, Cell Culture, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Over Expression

    6-AN displayed anti-HBV activity in an in vitro HBV infection model. PHH cells were infected with 2 × 10 3 genome equivalents/cell of HBV particles in the presence of 4% PEG8000 and then co-cultured with 6-AN (0 μM, 25 μM), ETV (0.5 μM) and 6-AN combined ETV for 10 days. (a) Cell culture supernatant were collected for HBsAg analysis via ELISA. 6-AN caused an obvious reduction of HBsAg level of secretion. (b) Western blot proved 6-AN significantly reduced HBsAg in cells. (c-d) 6-AN inhibited the level of total HBV RNAs (c) and 3.5-kb RNA (d) dose-dependently in PHH cells. (e) Northern blot proved that 6-AN not only reduced the 3.5-kb RNA, but also 2.4/2.1-kb RNA. (f-g) 6-AN treatment decreased the level of HBV core DNA in supernatant (f) and in cells (g). (h) Southern blot got a consistent decline. (i) 6-AN treatment showed a little degree reduction of HBV cccDNA level. Results are expressed as the average of four independent experiments ( n = 4 per group). The mean value ± standard error is indicated. (* P
    Figure Legend Snippet: 6-AN displayed anti-HBV activity in an in vitro HBV infection model. PHH cells were infected with 2 × 10 3 genome equivalents/cell of HBV particles in the presence of 4% PEG8000 and then co-cultured with 6-AN (0 μM, 25 μM), ETV (0.5 μM) and 6-AN combined ETV for 10 days. (a) Cell culture supernatant were collected for HBsAg analysis via ELISA. 6-AN caused an obvious reduction of HBsAg level of secretion. (b) Western blot proved 6-AN significantly reduced HBsAg in cells. (c-d) 6-AN inhibited the level of total HBV RNAs (c) and 3.5-kb RNA (d) dose-dependently in PHH cells. (e) Northern blot proved that 6-AN not only reduced the 3.5-kb RNA, but also 2.4/2.1-kb RNA. (f-g) 6-AN treatment decreased the level of HBV core DNA in supernatant (f) and in cells (g). (h) Southern blot got a consistent decline. (i) 6-AN treatment showed a little degree reduction of HBV cccDNA level. Results are expressed as the average of four independent experiments ( n = 4 per group). The mean value ± standard error is indicated. (* P

    Techniques Used: Activity Assay, In Vitro, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Northern Blot, Southern Blot

    24) Product Images from "Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells"

    Article Title: Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl389

    HBx interacts with ERα in vitro and in vivo . ( A ) Interaction of HBx with ERα in vitro . A GST pull-down assay was performed using 35 S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. ( B ) Interaction of HBx with ERα in vivo . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). ( C ) Interaction of endogenous HBx with ERα in vivo . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). ( D ) Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 µg ERα, 4 µg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 µg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. ( E ) Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E 2 for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. ( F ) Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using 35 S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. ( G ) Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and 35 S-labeled full-length ERα (1–595), ERα (1–185), ERα (180–282), ERα (282–595), ERα (302–595) or ERΔ5. Schematic diagram of the ERα deletion constructs used is shown at the top.
    Figure Legend Snippet: HBx interacts with ERα in vitro and in vivo . ( A ) Interaction of HBx with ERα in vitro . A GST pull-down assay was performed using 35 S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. ( B ) Interaction of HBx with ERα in vivo . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). ( C ) Interaction of endogenous HBx with ERα in vivo . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). ( D ) Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 µg ERα, 4 µg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 µg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. ( E ) Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E 2 for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. ( F ) Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using 35 S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. ( G ) Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and 35 S-labeled full-length ERα (1–595), ERα (1–185), ERα (180–282), ERα (282–595), ERα (302–595) or ERΔ5. Schematic diagram of the ERα deletion constructs used is shown at the top.

    Techniques Used: In Vitro, In Vivo, Pull Down Assay, Labeling, SDS Page, Autoradiography, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Microscopy, Staining, Construct, Binding Assay, Purification

    25) Product Images from "Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields"

    Article Title: Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01112

    Assessments of direct and Fc-mediated effects of anti-HER2 Fc variants against breast cancer cells. (A) Effects of anti-HER2 antibody variants on the proliferation of trastuzumab-sensitive (BT-474, SK-BR-3), trastuzumab-resistant (HCC1954) and triple-negative (MDA-MB-231) breast cancer cell lines. Anti-HER2 variants inhibited the proliferation of BT-474 and SK-BR-3 cells in a similar dose-dependent manner, but did not affect the proliferation of MDA-MB-231 or HCC1954 cells. Graphs represent an average of two experiments ± SD. (B) Human peripheral blood NK cell-mediated ADCC of BT-474 cancer cells induced by anti-HER2 variants measured by LDH release. Graphs are representative of independent experiments with three different human NK cell donors; data were normalized to minimal and maximal cell lysis. Error bars represent SEM values from technical replicates. N/D: not detected. (C) Effective concentration [(EC50) nM] measurements of ADCC by three human NK cell donors. (D) NK cell-mediated ADCC (measured by LDH release) of HER2 low (MDA-MB-231) and HER2 high (BT-474) breast cancer cells induced by anti-HER2 variants. The flow cytometric histograms on the left depict HER2 expression levels in MDA-MB-231 (top) and BT-474 (bottom) compared to unstained cells or cells stained with isotype control mAb. The graphs represent total cell killing levels of MDA-MB-231 cells (top) and BT-474 cells (bottom) mediated by NK cells from two different donors (Donors 4 and 5) at different concentrations of anti-HER2 variants.
    Figure Legend Snippet: Assessments of direct and Fc-mediated effects of anti-HER2 Fc variants against breast cancer cells. (A) Effects of anti-HER2 antibody variants on the proliferation of trastuzumab-sensitive (BT-474, SK-BR-3), trastuzumab-resistant (HCC1954) and triple-negative (MDA-MB-231) breast cancer cell lines. Anti-HER2 variants inhibited the proliferation of BT-474 and SK-BR-3 cells in a similar dose-dependent manner, but did not affect the proliferation of MDA-MB-231 or HCC1954 cells. Graphs represent an average of two experiments ± SD. (B) Human peripheral blood NK cell-mediated ADCC of BT-474 cancer cells induced by anti-HER2 variants measured by LDH release. Graphs are representative of independent experiments with three different human NK cell donors; data were normalized to minimal and maximal cell lysis. Error bars represent SEM values from technical replicates. N/D: not detected. (C) Effective concentration [(EC50) nM] measurements of ADCC by three human NK cell donors. (D) NK cell-mediated ADCC (measured by LDH release) of HER2 low (MDA-MB-231) and HER2 high (BT-474) breast cancer cells induced by anti-HER2 variants. The flow cytometric histograms on the left depict HER2 expression levels in MDA-MB-231 (top) and BT-474 (bottom) compared to unstained cells or cells stained with isotype control mAb. The graphs represent total cell killing levels of MDA-MB-231 cells (top) and BT-474 cells (bottom) mediated by NK cells from two different donors (Donors 4 and 5) at different concentrations of anti-HER2 variants.

    Techniques Used: Multiple Displacement Amplification, Lysis, Concentration Assay, Flow Cytometry, Expressing, Staining

    26) Product Images from "Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus"

    Article Title: Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01734-18

    NVR 3-778 targets HBV core protein and inhibits viral replication. (A) Chemical structure of NVR 3-778. (B) Purified recombinant HBV core protein in the absence or presence of NVR 3-778. NVR 3-778 was incubated with recombinant core protein (1.2:1 compound-to-core monomer ratio) in buffer containing 0.15 M NaCl at room temperature overnight prior to staining and imaging by electron microscopy. (C, D) The effect of NVR 3-778 on intracellular rcDNA, extracellular HBV DNA, or cell viability (C) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (D) was determined upon treating HepG2.2.15 cells with increasing concentrations of NVR 3-778. (E, F) The effect of TFV on intracellular rcDNA, extracellular HBV DNA, or cell viability (E) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (F) was determined upon treating HepG2.2.15 cells with increasing concentrations of TFV. Secreted HBV DNA and secreted HBV RNA levels were determined from the supernatant of HepG2.2.15 cells. Intracellular encapsidated rcDNA and pgRNA levels were determined upon NP-40 lysis of cells and by using S7 nuclease to remove nonencapsidated nucleic acids. Cell viability was determined by measuring ATP levels using the CellTiter-Glo assay. Data points are mean values from at least three independent experiments, with standard deviations shown as error bars.
    Figure Legend Snippet: NVR 3-778 targets HBV core protein and inhibits viral replication. (A) Chemical structure of NVR 3-778. (B) Purified recombinant HBV core protein in the absence or presence of NVR 3-778. NVR 3-778 was incubated with recombinant core protein (1.2:1 compound-to-core monomer ratio) in buffer containing 0.15 M NaCl at room temperature overnight prior to staining and imaging by electron microscopy. (C, D) The effect of NVR 3-778 on intracellular rcDNA, extracellular HBV DNA, or cell viability (C) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (D) was determined upon treating HepG2.2.15 cells with increasing concentrations of NVR 3-778. (E, F) The effect of TFV on intracellular rcDNA, extracellular HBV DNA, or cell viability (E) or on intracellular encapsidated pgRNA- or extracellular HBV RNA-containing particles (F) was determined upon treating HepG2.2.15 cells with increasing concentrations of TFV. Secreted HBV DNA and secreted HBV RNA levels were determined from the supernatant of HepG2.2.15 cells. Intracellular encapsidated rcDNA and pgRNA levels were determined upon NP-40 lysis of cells and by using S7 nuclease to remove nonencapsidated nucleic acids. Cell viability was determined by measuring ATP levels using the CellTiter-Glo assay. Data points are mean values from at least three independent experiments, with standard deviations shown as error bars.

    Techniques Used: Purification, Recombinant, Incubation, Staining, Imaging, Electron Microscopy, Lysis, Glo Assay

    27) Product Images from "Performance of the Cobas® Influenza A/B Assay for Rapid Pcr-Based Detection of Influenza Compared to Prodesse ProFlu+ and Viral Culture"

    Article Title: Performance of the Cobas® Influenza A/B Assay for Rapid Pcr-Based Detection of Influenza Compared to Prodesse ProFlu+ and Viral Culture

    Journal: European Journal of Microbiology & Immunology

    doi: 10.1556/1886.2015.00046

    cobas ® Influenza A/B assay workflow. A sample is collected directly into a Liat Tube (A). After the tube is capped, the analyzer scans the tube barcode (B), and the tube is inserted in the analyzer (C). Then, the analyzer automatically performs all the nucleic acid extraction and amplification steps and reports results in ~20 min
    Figure Legend Snippet: cobas ® Influenza A/B assay workflow. A sample is collected directly into a Liat Tube (A). After the tube is capped, the analyzer scans the tube barcode (B), and the tube is inserted in the analyzer (C). Then, the analyzer automatically performs all the nucleic acid extraction and amplification steps and reports results in ~20 min

    Techniques Used: Amplification

    28) Product Images from "Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience"

    Article Title: Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience

    Journal: Advances in Therapy

    doi: 10.1007/s12325-017-0612-x

    Mechanisms of rituximab-mediated cell death. Rituximab-coated B cells are killed by at least four different mechanisms: (1) binding of rituximab to CD20 on the B-cell surface causes activation of the complement cascade, which generates the membrane attack complex (MAC), which can directly induce B-cell lysis by complement-dependent cytotoxicity (CDC). (2) Binding of rituximab allows interaction with natural killer (NK) cells via Fc receptors (FcRs) III, which leads to antibody-dependent cellular cytotoxicity (ADCC). (3) The Fc portion of rituximab and the deposited complement fragments allow recognition by both FcRs and complement receptors on macrophages, which leads to phagocytosis and ADCC. (4) The crosslinking of several molecules of rituximab and CD20 in the lipid raft determines the interaction of these complexes with elements of a signaling pathway involving Src kinases that mediate direct apoptosis. FCR Fc receptor, FCγR Fcγ receptor. (Republished with permission of the American Society of Hematology from Jaglowski et al. [ 166 ]; permission conveyed through the Copyright Clearance Center)
    Figure Legend Snippet: Mechanisms of rituximab-mediated cell death. Rituximab-coated B cells are killed by at least four different mechanisms: (1) binding of rituximab to CD20 on the B-cell surface causes activation of the complement cascade, which generates the membrane attack complex (MAC), which can directly induce B-cell lysis by complement-dependent cytotoxicity (CDC). (2) Binding of rituximab allows interaction with natural killer (NK) cells via Fc receptors (FcRs) III, which leads to antibody-dependent cellular cytotoxicity (ADCC). (3) The Fc portion of rituximab and the deposited complement fragments allow recognition by both FcRs and complement receptors on macrophages, which leads to phagocytosis and ADCC. (4) The crosslinking of several molecules of rituximab and CD20 in the lipid raft determines the interaction of these complexes with elements of a signaling pathway involving Src kinases that mediate direct apoptosis. FCR Fc receptor, FCγR Fcγ receptor. (Republished with permission of the American Society of Hematology from Jaglowski et al. [ 166 ]; permission conveyed through the Copyright Clearance Center)

    Techniques Used: Binding Assay, Activation Assay, Lysis

    29) Product Images from "Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT"

    Article Title: Inventory of telomerase components in human cells reveals multiple subpopulations of hTR and hTERT

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku560

    Analysis of hTR, hTERT and telomerase activity in the hTERT IP samples. ( a ) hTERT was immunoprecipitated from HEK 293T cell lysate and eluted from beads with excess amounts of the corresponding peptide antigen as described in ‘Materials and Methods’ section. hTR in the input (0.67%), flow through (0.67%) and elution (40%) fractions was examined by northern blot, with H1 RNA serving as an internal control. Western blot with Abcam ab32020 was performed on the input (0.1%), flow through (0.1%) and elution (6%) fractions, with β-actin serving as an internal control. ( b ) Telomerase activity in the input (1%), flow through (1%) and elution (7.5%) fractions was examined by the direct enzyme assay as described in ‘Materials and Methods’ section. (1, 2) Duplicate repeats of experiment. +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. LC, labeled unextended primer, serving as a loading control. ( c ) Summary of hTR levels, Abcam ab32020 western signals and telomerase activity levels present in flow through and elution fractions, as normalized to input levels, in the specific experiment shown in (a) and (b). Note that the telomerase activity in the elution (∼69% input) is higher than that lost from input to flow through (∼35% input), probably because cellular factors present in input/flow through but not elution are inhibiting telomerase activity. Replicates of this experiment in HEK 293T and HeLa cells are summarized in Table 2 .
    Figure Legend Snippet: Analysis of hTR, hTERT and telomerase activity in the hTERT IP samples. ( a ) hTERT was immunoprecipitated from HEK 293T cell lysate and eluted from beads with excess amounts of the corresponding peptide antigen as described in ‘Materials and Methods’ section. hTR in the input (0.67%), flow through (0.67%) and elution (40%) fractions was examined by northern blot, with H1 RNA serving as an internal control. Western blot with Abcam ab32020 was performed on the input (0.1%), flow through (0.1%) and elution (6%) fractions, with β-actin serving as an internal control. ( b ) Telomerase activity in the input (1%), flow through (1%) and elution (7.5%) fractions was examined by the direct enzyme assay as described in ‘Materials and Methods’ section. (1, 2) Duplicate repeats of experiment. +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. LC, labeled unextended primer, serving as a loading control. ( c ) Summary of hTR levels, Abcam ab32020 western signals and telomerase activity levels present in flow through and elution fractions, as normalized to input levels, in the specific experiment shown in (a) and (b). Note that the telomerase activity in the elution (∼69% input) is higher than that lost from input to flow through (∼35% input), probably because cellular factors present in input/flow through but not elution are inhibiting telomerase activity. Replicates of this experiment in HEK 293T and HeLa cells are summarized in Table 2 .

    Techniques Used: Activity Assay, Immunoprecipitation, Flow Cytometry, Northern Blot, Western Blot, Enzymatic Assay, Labeling

    Measurement of the specific activity of endogenous telomerase and of super-telomerase. ( a ) Quantification of the hTR molecules co-immunoprecipitated with hTERT. The number of endogenous or overexpressed hTR molecules in the corresponding elution samples was measured by comparing northern blot signals obtained from a titration of Std hTR to that from hTR present in the indicated volumes of elutions. ( b ) Quantification of activity of telomerase eluted from antibody beads by the direct assay (see ‘Materials and Methods’ section). (1, 2, 3) Triplicate repeats of experiment. +0, labeled unextended primer, which also served as a loading control (LC). +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. Signals of extension products on the gel were compared to that of the 18-mer LC to calculate the absolute radioactivity of the extension products. Given the number of hTR molecules in the elution samples measured in (a), the specific activity (nt incorporated per telomerase per minute) was then calculated. ( c ) Telomerase activity as a function of dGTP concentration shown as a Lineweaver–Burk plot. Each point is the average of two technical replicates. One of two biological replicates is shown. For super-telomerase, V max = 57 ± 10 nt incorporated per telomerase monomer per minute, K m = 17 ± 3 μM; for endogenous telomerase, V max = 59 ± 8 nt incorporated per telomerase monomer per minute, K m = 16 ± 2 μM (mean ± SD, n = 2 biological replicates.)
    Figure Legend Snippet: Measurement of the specific activity of endogenous telomerase and of super-telomerase. ( a ) Quantification of the hTR molecules co-immunoprecipitated with hTERT. The number of endogenous or overexpressed hTR molecules in the corresponding elution samples was measured by comparing northern blot signals obtained from a titration of Std hTR to that from hTR present in the indicated volumes of elutions. ( b ) Quantification of activity of telomerase eluted from antibody beads by the direct assay (see ‘Materials and Methods’ section). (1, 2, 3) Triplicate repeats of experiment. +0, labeled unextended primer, which also served as a loading control (LC). +2 and +4, size markers made by extending the DNA primer by two or four nucleotides. Signals of extension products on the gel were compared to that of the 18-mer LC to calculate the absolute radioactivity of the extension products. Given the number of hTR molecules in the elution samples measured in (a), the specific activity (nt incorporated per telomerase per minute) was then calculated. ( c ) Telomerase activity as a function of dGTP concentration shown as a Lineweaver–Burk plot. Each point is the average of two technical replicates. One of two biological replicates is shown. For super-telomerase, V max = 57 ± 10 nt incorporated per telomerase monomer per minute, K m = 17 ± 3 μM; for endogenous telomerase, V max = 59 ± 8 nt incorporated per telomerase monomer per minute, K m = 16 ± 2 μM (mean ± SD, n = 2 biological replicates.)

    Techniques Used: Activity Assay, Immunoprecipitation, Northern Blot, Titration, Labeling, Radioactivity, Concentration Assay

    Overexpression of either hTR or hTERT increases total telomerase activity in living cells. ( a ) hTR or hTERT overexpression in HEK 293T and HeLa cells was quantified by northern or western blot, respectively, with H1 RNA or β-actin as loading controls. ( b ) Quantification of telomerase activity as measured by direct telomerase assay using lysates from HEK 293T or HeLa cells untransfected or transfected with either hTR or hTERT expression vectors (error bars: SD, n = 3).
    Figure Legend Snippet: Overexpression of either hTR or hTERT increases total telomerase activity in living cells. ( a ) hTR or hTERT overexpression in HEK 293T and HeLa cells was quantified by northern or western blot, respectively, with H1 RNA or β-actin as loading controls. ( b ) Quantification of telomerase activity as measured by direct telomerase assay using lysates from HEK 293T or HeLa cells untransfected or transfected with either hTR or hTERT expression vectors (error bars: SD, n = 3).

    Techniques Used: Over Expression, Activity Assay, Northern Blot, Western Blot, Telomerase Assay, Transfection, Expressing

    30) Product Images from "Genome Sequence and Characterization of the Tsukamurella Bacteriophage TPA2 ▿ Bacteriophage TPA2 ▿ †"

    Article Title: Genome Sequence and Characterization of the Tsukamurella Bacteriophage TPA2 ▿ Bacteriophage TPA2 ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01938-10

    Circular map of the TPA2 genome. The arrows represent the putative genes and the directions in which they are transcribed. Modules are shaded in similar tones, and the outer circle indicates the genes encoded within the modules. Arrows represent the repeat
    Figure Legend Snippet: Circular map of the TPA2 genome. The arrows represent the putative genes and the directions in which they are transcribed. Modules are shaded in similar tones, and the outer circle indicates the genes encoded within the modules. Arrows represent the repeat

    Techniques Used:

    Genomic features of TPA2.
    Figure Legend Snippet: Genomic features of TPA2.

    Techniques Used:

    31) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    Journal: Journal of Virology

    doi: 10.1128/JVI.00798-18

    CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.
    Figure Legend Snippet: CsCl density gradient analysis of hepatitis B viral particles. (A and B) CsCl density gradient analysis of viral particles in patient sera. One hundred-microliter volumes of serum mixture from patients 37, 38, 14, and 35 (25 μl each) and 100 μl serum from patient 17 were separated by CsCl density gradient centrifugation (2 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 2.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral DNA in each fraction was extracted and detected by Southern blotting. (C to G) CsCl density gradient analysis of viral particles treated with detergent or anti-HBcAg antibody (Ab). Concentrated HepAD38 cell culture supernatant (250 μl each) (via ultrafiltration) was either mixed with anti-HBcAg antibody (10 μl) followed by incubation without (C) or with NP-40 (final concentration, 1%) (D) for 1 h at room temperature and 4 h on ice or treated with only NP-40 (G) and then fractionated by CsCl density gradient ultracentrifugation. Sera from CHB patient 46 either left untreated (E) or treated with NP-40 (final concentration, 1%) (F) were fractionated by CsCl density gradient ultracentrifugation. Viral DNA in each fraction was extracted and subjected to Southern blot analyses.

    Techniques Used: Gradient Centrifugation, Southern Blot, Cell Culture, Incubation, Concentration Assay

    32) Product Images from "PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma"

    Article Title: PI3Kδ inhibition causes feedback activation of PI3Kα in the ABC subtype of diffuse large B-cell lymphoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20864

    Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.
    Figure Legend Snippet: Feedback activation of PI3Kα following PI3Kδ inhibition depends on increased BCR signaling A. TMD8 was exposed over different time periods to CAL-101. Western blot indicates increased proximal BCR signaling following PI3Kδ inhibition. B. TMD8 and Ly10 were treated with 200nM CAL-101, 50nM Dasatinib (src inhibitor), 1000nM PRT062607 (Syk inhibitor) at the indicated time points and harvested at 2hr and 24hr. Results indicates that rebound PI3K reactivation following PI3Kδ inhibition is sensitive to Src and Syk inhibition. C. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with BCAP and probed for the indicated proteins. D. TMD8 and Ly10 were treated with CAL-101 over 0, 6 and 16hr. Cells were harvested, lysed with NP-40 lysis buffer, immunoprecipitated with CD19 and probed for the indicated proteins.

    Techniques Used: Activation Assay, Inhibition, Western Blot, Lysis, Immunoprecipitation

    33) Product Images from "Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact"

    Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036349

    Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.
    Figure Legend Snippet: Correlations between different markers for viral productivity in liver and serum. A-C show strong correlation in 19 liver biopsies between cccDNA levels and pgRNA (A) and S-RNA (B), and significant correlation between cccDNA and pgRNA/S-RNA ratio (C). D shows strong correlation between pgRNA and serum levels of HBV DNA (but lack of correlation within the HBeAg-negative subgroup). E shows relatively strong correlation between S-RNA and HBsAg in serum. Filled dots HBeAg positive, open dots HBeAg negative.

    Techniques Used:

    Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.
    Figure Legend Snippet: Levels of cccDNA and HBV RNA in vivo and in vitro. The cccDNA levels (A) and pgRNA per cccDNA (B), as well as pgRNA/cccDNA ratios (C) were higher in liver tissue from HBeAg-positive as compared with HBeAg-negative patients. In PLC/PRF/5 cells, the cccDNA PCR amplifies integrated HBV DNA (a segment containing the promoter for pgRNA). In these cells which contain multiple integrations of the S region, the pgRNA/S-RNA ratio was low (C). In Huh7.5 cells, the cccDNA levels, pgRNA per cccDNA and ratio between pgRNA and S-RNA were similar in cells transfected with HBV without or with mutations in the core promoter region, indicating that these mutations have low impact on pgRNA transcription.

    Techniques Used: In Vivo, In Vitro, Planar Chromatography, Polymerase Chain Reaction, Transfection

    34) Product Images from "Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells"

    Article Title: Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl389

    HBx interacts with ERα in vitro and in vivo . ( A ) Interaction of HBx with ERα in vitro . A GST pull-down assay was performed using 35 S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. ( B ) Interaction of HBx with ERα in vivo . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). ( C ) Interaction of endogenous HBx with ERα in vivo . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). ( D ) Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 µg ERα, 4 µg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 µg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. ( E ) Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E 2 for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. ( F ) Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using 35 S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. ( G ) Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and 35 S-labeled full-length ERα (1–595), ERα (1–185), ERα (180–282), ERα (282–595), ERα (302–595) or ERΔ5. Schematic diagram of the ERα deletion constructs used is shown at the top.
    Figure Legend Snippet: HBx interacts with ERα in vitro and in vivo . ( A ) Interaction of HBx with ERα in vitro . A GST pull-down assay was performed using 35 S-labeled ERα, and GST or GST-HBx. The bound proteins were subjected to SDS–PAGE followed by autoradiography. ( B ) Interaction of HBx with ERα in vivo . ERα and FLAG-tagged HBx or empty vector were co-transfected into HepG2 cells. Cell lysates were immunoprecipitated (IP) by anti-FLAG M2 monoclonal antibody (Sigma), and the precipitates were then immunoblotted (IB) with anti-ERα polyclonal antibody (Santa Cruz Biotech). ( C ) Interaction of endogenous HBx with ERα in vivo . Liver tissue extracts from an HBV positive patient were immunoprecipitated with either anti-ERα polyclonal antibody or preimmune control serum (Santa Cruz Biotech). The precipitates were analyzed by immunoblot using anti-HBx (Chemicon). ( D ) Effect of ERΔ5 on the interaction between HBx and ERα. HepG2 cells were co-transfected with 2 µg ERα, 4 µg FLAG-tagged HBx and increasing amounts of ERΔ5 (2 and 4 µg). Cell lysates were immunoprecipitated by anti-FLAG monoclonal antibody, and the precipitates were detected with anti-ERα polyclonal antibody. ( E ) Co-localization of HBx and ERα in HepG2 cells. Cells were transfected with EGFP-tagged HBx and RFP-tagged ERα or empty vector (RFP) as indicated, and were treated with 10 nM E 2 for 24 h. The images were captured by confocal immunofluorescence microscopy. HBx localization is shown with EGFP (green) and ERα is seen with RFP (red). The nuclei were stained with DAPI (blue). Co-localization of HBx with ERα is shown in merged images. ( F ) Mapping of the ERα interaction region in HBx. A GST pull-down assay was performed using 35 S-labeled ERα and GST-HBx(1-72), GST-HBx(73-120), GST-HBx(121-154), GST-HBx(1-143), GST-HBx(52-154) and full-length GST-HBx(1–154) or GST. Schematic diagram of the HBx deletion constructs used is shown at the top, the binding of ERα to different regions of HBx is demonstrated in the middle, and SDS–PAGE analysis of the purified GST-fusion proteins is shown at the bottom. Asterisks indicate the positions of the expected purified GST or GST-fusion proteins. ( G ) Mapping of the HBx interaction region in ERα. A GST pull-down assay was performed using full-length GST-HBx(1–154) or GST, and 35 S-labeled full-length ERα (1–595), ERα (1–185), ERα (180–282), ERα (282–595), ERα (302–595) or ERΔ5. Schematic diagram of the ERα deletion constructs used is shown at the top.

    Techniques Used: In Vitro, In Vivo, Pull Down Assay, Labeling, SDS Page, Autoradiography, Plasmid Preparation, Transfection, Immunoprecipitation, Immunofluorescence, Microscopy, Staining, Construct, Binding Assay, Purification

    The HBx deletion mutant abolishes HBx-induced repression of ERα transcriptional activity. ( A ) Luciferase reporter assays with the HBx deletion mutants. HepG2 cells were co-transfected with 0.2 µg of ERE-LUC, 50 ng of the expression plasmid for ERα and 1.0 µg of the expression vector for FLAG-tagged HBx or HBx(Δ73–120), in the presence or absence of 10 nM E 2 . ( B ) Western blotting showing expression of FLAG-tagged HBx and HBx(Δ73–120). Cells were transfected as in (A). Cell extracts were prepared from E 2 -treated cells, and equivalent amounts of each extract were detected with anti-FLAG or anti-GAPDH antibody. ( C ) The HBx deletion mutant abolishes the HBx–ERα interaction. HepG2 cells were co-transfected with the expression plasmid for ERα and the expression vector for FLAG-tagged HBx or HBx(Δ73–120). Cell lysates were immunoprecipitated by anti-FLAG, and the precipitates were probed with anti-ERα.
    Figure Legend Snippet: The HBx deletion mutant abolishes HBx-induced repression of ERα transcriptional activity. ( A ) Luciferase reporter assays with the HBx deletion mutants. HepG2 cells were co-transfected with 0.2 µg of ERE-LUC, 50 ng of the expression plasmid for ERα and 1.0 µg of the expression vector for FLAG-tagged HBx or HBx(Δ73–120), in the presence or absence of 10 nM E 2 . ( B ) Western blotting showing expression of FLAG-tagged HBx and HBx(Δ73–120). Cells were transfected as in (A). Cell extracts were prepared from E 2 -treated cells, and equivalent amounts of each extract were detected with anti-FLAG or anti-GAPDH antibody. ( C ) The HBx deletion mutant abolishes the HBx–ERα interaction. HepG2 cells were co-transfected with the expression plasmid for ERα and the expression vector for FLAG-tagged HBx or HBx(Δ73–120). Cell lysates were immunoprecipitated by anti-FLAG, and the precipitates were probed with anti-ERα.

    Techniques Used: Mutagenesis, Activity Assay, Luciferase, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

    HBx inhibits ERα-mediated transactivation function in hepatoma cells. ( A ) HepG2 cells were co-transfected with 0.2 µg of ERE-Luc, 50 ng of the expression plasmid for ERα and increasing amounts of the expression plasmid for FLAG-tagged HBx in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 was set as 1. ( B ) Immunoblotting showing the ERα and HBx levels in HepG2 cells. Cells were transfected as in (A). Whole cell extracts were prepared from the cells transfected with 2.0 µg of the expression plasmid for HBx in the presence of 10 nM E 2 , and were detected with anti-ERα (Santa Cruz Biotech), anti-FLAG (Sigma) or anti-GAPDH (Biogenesis) antibody. (C–E) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx, and 0.2 µg of C3-Luc ( C ), pS2-Luc ( D ) or pS2ΔERE-Luc ( E ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα and HBx in the absence of E 2 was set as 1.
    Figure Legend Snippet: HBx inhibits ERα-mediated transactivation function in hepatoma cells. ( A ) HepG2 cells were co-transfected with 0.2 µg of ERE-Luc, 50 ng of the expression plasmid for ERα and increasing amounts of the expression plasmid for FLAG-tagged HBx in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 was set as 1. ( B ) Immunoblotting showing the ERα and HBx levels in HepG2 cells. Cells were transfected as in (A). Whole cell extracts were prepared from the cells transfected with 2.0 µg of the expression plasmid for HBx in the presence of 10 nM E 2 , and were detected with anti-ERα (Santa Cruz Biotech), anti-FLAG (Sigma) or anti-GAPDH (Biogenesis) antibody. (C–E) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx, and 0.2 µg of C3-Luc ( C ), pS2-Luc ( D ) or pS2ΔERE-Luc ( E ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα and HBx in the absence of E 2 was set as 1.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    Treatment of hepatoma cells with the specific HDAC inhibitor TSA causes a drastic relieving of HBx-induced repression of ERα transactivation. HepG2 cells co-transfected with 0.2 µg of the ERE-Luc reporter, 50 ng of the expression vector for ERα, 1.0 µg of the expression plasmid for HBx. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 or 100 nM TSA as indicated. The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 and TSA was set as 1.
    Figure Legend Snippet: Treatment of hepatoma cells with the specific HDAC inhibitor TSA causes a drastic relieving of HBx-induced repression of ERα transactivation. HepG2 cells co-transfected with 0.2 µg of the ERE-Luc reporter, 50 ng of the expression vector for ERα, 1.0 µg of the expression plasmid for HBx. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 or 100 nM TSA as indicated. The luciferase activity obtained on transfection of ERE-Luc and ERα without exogenous HBx in the absence of E 2 and TSA was set as 1.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    HBx and ERΔ5 have additive effect on repression of specific ERα responsive gene transcription. (A–D) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx, 50 ng of the expression plasmid for ERΔ5, and 0.2 µg of ERE-Luc ( A ), C3-Luc ( B ), pS2-Luc ( C ) or pS2ΔERE-Luc ( D ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1. ( E ) HepG2 cells were co-transfected with 0.2 µg of pS2-Luc, 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx and 50 ng of the expression vector for ERΔ5. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 , 100 nM 4-hydroxytamoxifen (4-OHT) or 10 nM E 2 plus 100 nM 4-OHT. The luciferase activity obtained on transfection of pS2-Luc without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1.
    Figure Legend Snippet: HBx and ERΔ5 have additive effect on repression of specific ERα responsive gene transcription. (A–D) HepG2 cells were co-transfected with 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx, 50 ng of the expression plasmid for ERΔ5, and 0.2 µg of ERE-Luc ( A ), C3-Luc ( B ), pS2-Luc ( C ) or pS2ΔERE-Luc ( D ), in the absence or presence of 10 nM E 2 . The luciferase activity obtained on transfection of the respective luciferase reporter without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1. ( E ) HepG2 cells were co-transfected with 0.2 µg of pS2-Luc, 50 ng of the expression plasmid for ERα, 1.0 µg of the expression plasmid for FLAG-tagged HBx and 50 ng of the expression vector for ERΔ5. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E 2 , 100 nM 4-hydroxytamoxifen (4-OHT) or 10 nM E 2 plus 100 nM 4-OHT. The luciferase activity obtained on transfection of pS2-Luc without exogenous ERα, ERΔ5 and HBx in the absence of E 2 was set as 1.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    HBx forms a complex with ERα and HDAC1. ( A ) Association of HBx with HDAC1 in vitro . GST-HBx and GST were incubated with 35 S-labeled ERα, and a GST pull-down assay was then performed. ( B ) Association of HBx with ERα in vivo . HepG2 cells were transiently transfected with HA-tagged HDAC1 and FLAG-tagged HBx or control vector. Immunoprecipitation (IP) was performed using anti-FLAG monoclonal antibody; immunoblotting (IB) was performed with the indicated antibodies. ( C ) HBx interacts with both HDAC1 and ERα in vivo . HepG2 cells were co-transfected with ERα, HA-tagged HDAC1, and FLAG-tagged HBx or control vector. The cell extracts were immunoprecipitated with anti-FLAG monoclonal antibody followed by immunoblotting with the indicated antibodies. ( D ) HBx, ERα and HDAC1 forms a ternary complex. HepG2 cells were transfected as in (C). The cell extracts were immunoprecipitated with anti-FLAG antibody. Immune complexes were eluted with FLAG peptide and re-immunoprecipitated (re-IP) using anti-ERα polyclonal antibody and normal rabbit serum as a negative control. The resulting precipitates were resolved by SDS–PAGE followed by immunoblotting with the indicated antibodies.
    Figure Legend Snippet: HBx forms a complex with ERα and HDAC1. ( A ) Association of HBx with HDAC1 in vitro . GST-HBx and GST were incubated with 35 S-labeled ERα, and a GST pull-down assay was then performed. ( B ) Association of HBx with ERα in vivo . HepG2 cells were transiently transfected with HA-tagged HDAC1 and FLAG-tagged HBx or control vector. Immunoprecipitation (IP) was performed using anti-FLAG monoclonal antibody; immunoblotting (IB) was performed with the indicated antibodies. ( C ) HBx interacts with both HDAC1 and ERα in vivo . HepG2 cells were co-transfected with ERα, HA-tagged HDAC1, and FLAG-tagged HBx or control vector. The cell extracts were immunoprecipitated with anti-FLAG monoclonal antibody followed by immunoblotting with the indicated antibodies. ( D ) HBx, ERα and HDAC1 forms a ternary complex. HepG2 cells were transfected as in (C). The cell extracts were immunoprecipitated with anti-FLAG antibody. Immune complexes were eluted with FLAG peptide and re-immunoprecipitated (re-IP) using anti-ERα polyclonal antibody and normal rabbit serum as a negative control. The resulting precipitates were resolved by SDS–PAGE followed by immunoblotting with the indicated antibodies.

    Techniques Used: In Vitro, Incubation, Labeling, Pull Down Assay, In Vivo, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, SDS Page

    35) Product Images from "The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *"

    Article Title: The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.637694

    Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).
    Figure Legend Snippet: Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).

    Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Labeling, Diagnostic Assay, Clone Assay, Western Blot, Expressing, Fluorescence, Microscopy

    MyoB subcellular location in asexual blood and mosquito stages. A, microscopic analysis of live P. falciparum asexual blood stage parasites expressing GFP-tagged MyoB. Shown are blood stage parasites of increasing maturity from early schizogony (two to four nuclei; 30 h post-invasion) through to mature segmenter forms (44 h post-invasion) and free merozoites analyzed by fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. GFP fluorescence was not detected in trophozoites (data not shown) and early schizont stages, and was first apparent in mature multinucleate schizonts as a number of single dots (40 h post-invasion). Following cytokinesis, a single dot was present associated with each nucleus at what appeared to be the apical end of the cell. The images, merged with the differential interference contrast ( DIC ) image, are shown in the right panel. Scale bar, 2 μm. B, immunofluorescence of MyoB-HA in P. knowlesi schizonts. The epitope is detected using a specific antibody and an AlexaFluor 488 secondary antibody. Nuclei are labeled with DAPI; the green , blue, and differential interference contrast-merged images are also shown. Scale bar, 2 μm. C, expression of MyoB-GFP in the three invasive stages: schizonts (merozoites), ookinetes, and sporozoites of P. berghei . GFP is detected by green fluorescence, and nuclei ( blue ) were labeled with Hoechst dye. The merged and differential interference contrast images are also shown. The white arrows indicate expression of MyoB-GFP at the apical end of the parasites. Scale bar, 5 μm. D, expression of MyoB-GFP in liver stage schizonts of P. berghei , 55 h after invasion by a sporozoite. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are shown in the right panel. Scale bar, 10 μm. E, schematic showing the three invasive stages of Plasmodium , merozoite, ookinete, and sporozoite. The approximate length from anterior to posterior is shown.
    Figure Legend Snippet: MyoB subcellular location in asexual blood and mosquito stages. A, microscopic analysis of live P. falciparum asexual blood stage parasites expressing GFP-tagged MyoB. Shown are blood stage parasites of increasing maturity from early schizogony (two to four nuclei; 30 h post-invasion) through to mature segmenter forms (44 h post-invasion) and free merozoites analyzed by fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. GFP fluorescence was not detected in trophozoites (data not shown) and early schizont stages, and was first apparent in mature multinucleate schizonts as a number of single dots (40 h post-invasion). Following cytokinesis, a single dot was present associated with each nucleus at what appeared to be the apical end of the cell. The images, merged with the differential interference contrast ( DIC ) image, are shown in the right panel. Scale bar, 2 μm. B, immunofluorescence of MyoB-HA in P. knowlesi schizonts. The epitope is detected using a specific antibody and an AlexaFluor 488 secondary antibody. Nuclei are labeled with DAPI; the green , blue, and differential interference contrast-merged images are also shown. Scale bar, 2 μm. C, expression of MyoB-GFP in the three invasive stages: schizonts (merozoites), ookinetes, and sporozoites of P. berghei . GFP is detected by green fluorescence, and nuclei ( blue ) were labeled with Hoechst dye. The merged and differential interference contrast images are also shown. The white arrows indicate expression of MyoB-GFP at the apical end of the parasites. Scale bar, 5 μm. D, expression of MyoB-GFP in liver stage schizonts of P. berghei , 55 h after invasion by a sporozoite. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are shown in the right panel. Scale bar, 10 μm. E, schematic showing the three invasive stages of Plasmodium , merozoite, ookinete, and sporozoite. The approximate length from anterior to posterior is shown.

    Techniques Used: Expressing, Fluorescence, Microscopy, Labeling, Immunofluorescence

    PfMLC-B colocalizes in the cell with MyoB, binds to MyoB in vivo, and its C-terminal domain binds to the MyoB C-terminal sequence in vitro . A, structural model of amino acids 508–645 of PfMLC-B. The protein backbone is shown as a green ribbon , with a space-fill model of the structure overlaid. B, panel i, MLC-B-GFP subcellular location in a P. falciparum schizont. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are also shown. Scale bar, 2 μm. Panel ii, MLC-HA localization to the apex of merozoites in a P. knowlesi schizont. The HA epitope was detected using a specific antibody, followed by a species-specific AlexaFluor 488-labeled secondary antibody. Nuclei ( blue ) were detected using DAPI. Merged images are also shown. Scale bar, 2 μm. Panel iii, indirect immunofluorescence of P. falciparum schizont to determine colocalization of MLC-B-GFP ( green ) with MyoB ( red ). Nuclei were counterstained with DAPI ( blue ). The merged images are also shown. Scale bar, 2 μm. C, analysis of proteins affinity-purified with GFP-Trap from lysates of parasites expressing either PfMyoB-GFP or PfMLC-B-GFP by SDS-PAGE, trypsin digestion, and LC-MS/MS. Following subtraction of the list of proteins detected in control experiments, the lists of proteins detected in the two preparations were compared. Two proteins, MyoB and MLC-B, were in common. D, analysis of the binding of the C-terminal domain of PfMLC-B to peptides derived from the sequence at the C terminus of MyoB by either circular dichroism ( panels i and ii ) or thermal unfolding in the presence of peptides based on the MyoB amino acid sequences, residues Ile 763 to Arg 775 and Asn 780 to His 800 ( panel iii ). E, alignment of the neck regions of PfMyoA, PfMyoB, and TgMyoA showing confirmed ( red ) and speculated ( blue ) light-chain binding regions.
    Figure Legend Snippet: PfMLC-B colocalizes in the cell with MyoB, binds to MyoB in vivo, and its C-terminal domain binds to the MyoB C-terminal sequence in vitro . A, structural model of amino acids 508–645 of PfMLC-B. The protein backbone is shown as a green ribbon , with a space-fill model of the structure overlaid. B, panel i, MLC-B-GFP subcellular location in a P. falciparum schizont. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are also shown. Scale bar, 2 μm. Panel ii, MLC-HA localization to the apex of merozoites in a P. knowlesi schizont. The HA epitope was detected using a specific antibody, followed by a species-specific AlexaFluor 488-labeled secondary antibody. Nuclei ( blue ) were detected using DAPI. Merged images are also shown. Scale bar, 2 μm. Panel iii, indirect immunofluorescence of P. falciparum schizont to determine colocalization of MLC-B-GFP ( green ) with MyoB ( red ). Nuclei were counterstained with DAPI ( blue ). The merged images are also shown. Scale bar, 2 μm. C, analysis of proteins affinity-purified with GFP-Trap from lysates of parasites expressing either PfMyoB-GFP or PfMLC-B-GFP by SDS-PAGE, trypsin digestion, and LC-MS/MS. Following subtraction of the list of proteins detected in control experiments, the lists of proteins detected in the two preparations were compared. Two proteins, MyoB and MLC-B, were in common. D, analysis of the binding of the C-terminal domain of PfMLC-B to peptides derived from the sequence at the C terminus of MyoB by either circular dichroism ( panels i and ii ) or thermal unfolding in the presence of peptides based on the MyoB amino acid sequences, residues Ile 763 to Arg 775 and Asn 780 to His 800 ( panel iii ). E, alignment of the neck regions of PfMyoA, PfMyoB, and TgMyoA showing confirmed ( red ) and speculated ( blue ) light-chain binding regions.

    Techniques Used: In Vivo, Sequencing, In Vitro, Fluorescence, Labeling, Immunofluorescence, Affinity Purification, Expressing, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Derivative Assay

    Generation of PkMyoB-HA and PbMyoB-GFP and detection of myob throughout the P. berghei life cycle. A, diagram representing C-terminal tagging of Pkmyob with a triple HA tag. Primers shown ( arrows ) were used to amplify the region of homology (6 and 7) for diagnostic PCR of WT parasite sequences (8 and 9) or parasites where integration had taken place (8 and 10). B, diagnostic PCR on genomic DNA showing integration of PkMyoB-HA into the myob locus in clone c1 with parental H.1 DNA as a control. C, Western blot with an anti-HA antibody on parental PkH.1 and PkMyoB-HA parasite lysates. In PkMyoB-HA parasites, a protein of ∼90 kDa is detected. The same blot was probed with an antibody against BiP to demonstrate equivalent sample loading. D, quantitative RT-PCR to show mRNA expression of Pbmyob during the parasite life cycle. Arginyl-tRNA synthetase and hsp70 were used as endogenous controls for normalization. Bars, three biological replicates, each ± S.E. AS, all asexual blood stages; Sch, schizonts; NG, nonactivated gametocytes; AG, activated gametocytes; Ook, ookinetes; Spz, sporozoites. E, diagram representing C-terminal tagging of Pb MyoB with GFP by single homologous recombination into endogenous myob gene, showing primers 11 and 12 to amplify the region of homology, and primers 13 and 14 to detect integration. F, confirmation of integration by PCR of Pbmyob-gfp using primer pair 13 and 14. G, Western blot of extracts of parasites expressing Pb MyoB-GFP or GFP.
    Figure Legend Snippet: Generation of PkMyoB-HA and PbMyoB-GFP and detection of myob throughout the P. berghei life cycle. A, diagram representing C-terminal tagging of Pkmyob with a triple HA tag. Primers shown ( arrows ) were used to amplify the region of homology (6 and 7) for diagnostic PCR of WT parasite sequences (8 and 9) or parasites where integration had taken place (8 and 10). B, diagnostic PCR on genomic DNA showing integration of PkMyoB-HA into the myob locus in clone c1 with parental H.1 DNA as a control. C, Western blot with an anti-HA antibody on parental PkH.1 and PkMyoB-HA parasite lysates. In PkMyoB-HA parasites, a protein of ∼90 kDa is detected. The same blot was probed with an antibody against BiP to demonstrate equivalent sample loading. D, quantitative RT-PCR to show mRNA expression of Pbmyob during the parasite life cycle. Arginyl-tRNA synthetase and hsp70 were used as endogenous controls for normalization. Bars, three biological replicates, each ± S.E. AS, all asexual blood stages; Sch, schizonts; NG, nonactivated gametocytes; AG, activated gametocytes; Ook, ookinetes; Spz, sporozoites. E, diagram representing C-terminal tagging of Pb MyoB with GFP by single homologous recombination into endogenous myob gene, showing primers 11 and 12 to amplify the region of homology, and primers 13 and 14 to detect integration. F, confirmation of integration by PCR of Pbmyob-gfp using primer pair 13 and 14. G, Western blot of extracts of parasites expressing Pb MyoB-GFP or GFP.

    Techniques Used: Diagnostic Assay, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Expressing, Homologous Recombination

    PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.
    Figure Legend Snippet: PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.

    Techniques Used: Western Blot, SDS Page

    PfMyoB-GFP remains at the anterior of the merozoite during invasion of the host cell. MyoB-GFP-expressing P. falciparum parasites were fixed during various stages of invasion, and then MyoB-GFP ( green ) was revealed using rabbit anti-GFP antibodies, RON4 ( red ) was detected using mAb 24C6, and nuclei were stained with DAPI ( blue ). Merged images, including the differential interference contrast, are shown, as well as a schematic of the invasion stage in which the moving junction is shown by the blue arrowheads , the extracellular merozoite is gray , and the intracellular parasite is denoted by a dotted line and is uncolored. The invasion steps have been divided into initial attachment, followed by early and late stages of invasion as well as the final steps of invasion with the release of the remnant junction and formation of the ring stage. Scale bar, 2 μm.
    Figure Legend Snippet: PfMyoB-GFP remains at the anterior of the merozoite during invasion of the host cell. MyoB-GFP-expressing P. falciparum parasites were fixed during various stages of invasion, and then MyoB-GFP ( green ) was revealed using rabbit anti-GFP antibodies, RON4 ( red ) was detected using mAb 24C6, and nuclei were stained with DAPI ( blue ). Merged images, including the differential interference contrast, are shown, as well as a schematic of the invasion stage in which the moving junction is shown by the blue arrowheads , the extracellular merozoite is gray , and the intracellular parasite is denoted by a dotted line and is uncolored. The invasion steps have been divided into initial attachment, followed by early and late stages of invasion as well as the final steps of invasion with the release of the remnant junction and formation of the ring stage. Scale bar, 2 μm.

    Techniques Used: Expressing, Staining

    36) Product Images from "The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *"

    Article Title: The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.637694

    Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).
    Figure Legend Snippet: Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).

    Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Labeling, Diagnostic Assay, Clone Assay, Western Blot, Expressing, Fluorescence, Microscopy

    PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.
    Figure Legend Snippet: PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.

    Techniques Used: Western Blot, SDS Page

    37) Product Images from "Analytical biochemistry of DNA-protein assemblies from crude cell extracts"

    Article Title: Analytical biochemistry of DNA-protein assemblies from crude cell extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm490

    Affinity purification of the DNA ends-binding proteins. The proteins were purified from HeLa nuclear extracts using the PCB-TetO target sequence immobilized on beads (lanes 2 and 4) and without oligonucleotide on beads as control (lane 3). ( A ) The chromatographic slurry was either treated with detergents (lane 2) or irradiated (lanes 3 and 4), then the released proteins were separated on an 8% SDS-PAGE gel and stained with Sypro Ruby protein gel stain. ( B ) The purification product obtained after irradiation was analyzed by western blot with antibodies against the DNA ends-binding proteins as indicated. MW, molecular weight markers.
    Figure Legend Snippet: Affinity purification of the DNA ends-binding proteins. The proteins were purified from HeLa nuclear extracts using the PCB-TetO target sequence immobilized on beads (lanes 2 and 4) and without oligonucleotide on beads as control (lane 3). ( A ) The chromatographic slurry was either treated with detergents (lane 2) or irradiated (lanes 3 and 4), then the released proteins were separated on an 8% SDS-PAGE gel and stained with Sypro Ruby protein gel stain. ( B ) The purification product obtained after irradiation was analyzed by western blot with antibodies against the DNA ends-binding proteins as indicated. MW, molecular weight markers.

    Techniques Used: Affinity Purification, Binding Assay, Purification, Sequencing, Irradiation, SDS Page, Staining, Western Blot, Molecular Weight

    38) Product Images from "CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells"

    Article Title: CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000755

    Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells. C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10 6 /ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) ex vivo or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10 4 CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p
    Figure Legend Snippet: Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells. C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10 6 /ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) ex vivo or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10 4 CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Staining, Ex Vivo, Incubation

    CD83 is upregulated on activated B cells. C57BL/6 mice derived spleen cells (2×10 6 /ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10 4 CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10 6 purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.
    Figure Legend Snippet: CD83 is upregulated on activated B cells. C57BL/6 mice derived spleen cells (2×10 6 /ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10 4 CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10 6 purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.

    Techniques Used: Mouse Assay, Derivative Assay, Staining, Expressing, Fluorescence, Dot Blot, Purification, SDS Page, Western Blot

    39) Product Images from "The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models"

    Article Title: The Antitumor Activity of IMGN529, a CD37-Targeting Antibody-Drug Conjugate, Is Potentiated by Rituximab in Non-Hodgkin Lymphoma Models

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2017.06.001

    ADCC, but not CDC, is augmented by the combination of IMGN529 and rituximab. ADCC assays were performed by incubating target U-2932 (A) or SU-DHL-4 (B) cells with human NK effector cells at E/T ratio of 3:1 or 4:1 and measuring LDH release. Cells were exposed to increasing concentrations of IMGN529 in the presence or absence of varying concentrations of rituximab or control IgG, as indicated. Percent specific lysis was calculated, and data shown are the mean of three experiments. (C) CDC activity against U-2932 cells ( left panel ) and SU-DHL-4 cells ( right panel ) was determined following incubating cells for 2 hours with increasing concentrations of rituximab +/− 5 μg/mL of IMGN529 or IgG1-SMCC-DM1 in the presence of human complement. Cell viability was assessed by alamarBlue assay.
    Figure Legend Snippet: ADCC, but not CDC, is augmented by the combination of IMGN529 and rituximab. ADCC assays were performed by incubating target U-2932 (A) or SU-DHL-4 (B) cells with human NK effector cells at E/T ratio of 3:1 or 4:1 and measuring LDH release. Cells were exposed to increasing concentrations of IMGN529 in the presence or absence of varying concentrations of rituximab or control IgG, as indicated. Percent specific lysis was calculated, and data shown are the mean of three experiments. (C) CDC activity against U-2932 cells ( left panel ) and SU-DHL-4 cells ( right panel ) was determined following incubating cells for 2 hours with increasing concentrations of rituximab +/− 5 μg/mL of IMGN529 or IgG1-SMCC-DM1 in the presence of human complement. Cell viability was assessed by alamarBlue assay.

    Techniques Used: Lysis, Activity Assay, Alamar Blue Assay

    40) Product Images from "Type I interferon signaling is required for activation of the inflammasome during Francisella infection"

    Article Title: Type I interferon signaling is required for activation of the inflammasome during Francisella infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20062665

    Type I IFN signaling is necessary for activation of the inflammasome during F. novicida and L. monocytogenes infections but not upon ATP treatment. (A) WT, IFNR −/− , and ASC −/− BMMs uninfected (Un) or infected with mglA (m) or WT F. novicida (W) were lysed at 9 h PI. Caspase-1 processing was visualized by detection of the p20 subunit only in WT macrophages infected with WT F. novicida . (B and C) IL-1β (B) and -18 (C) were quantified by ELISA in the supernatant of preactivated BMMs. Similar levels were detected in WT and IFNR −/− BMMs treated for 3 h with 5 mM ATP (left), whereas high levels were only detected in WT macrophages upon infection with F. novicida for 5 h (right). (D) Cell death (left) was strongly reduced in IFNR −/− compared with WT BMMs at 6 h PI with L. monocytogenes . Similarly, IL-1β (middle) and -18 (right) levels were lower in activated BMMs infected for 2.5 h with L. monocytogenes at the indicated MOI.
    Figure Legend Snippet: Type I IFN signaling is necessary for activation of the inflammasome during F. novicida and L. monocytogenes infections but not upon ATP treatment. (A) WT, IFNR −/− , and ASC −/− BMMs uninfected (Un) or infected with mglA (m) or WT F. novicida (W) were lysed at 9 h PI. Caspase-1 processing was visualized by detection of the p20 subunit only in WT macrophages infected with WT F. novicida . (B and C) IL-1β (B) and -18 (C) were quantified by ELISA in the supernatant of preactivated BMMs. Similar levels were detected in WT and IFNR −/− BMMs treated for 3 h with 5 mM ATP (left), whereas high levels were only detected in WT macrophages upon infection with F. novicida for 5 h (right). (D) Cell death (left) was strongly reduced in IFNR −/− compared with WT BMMs at 6 h PI with L. monocytogenes . Similarly, IL-1β (middle) and -18 (right) levels were lower in activated BMMs infected for 2.5 h with L. monocytogenes at the indicated MOI.

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

    Type I IFN induction and signaling is required for F. novicida –mediated but not for S. typhimurium –mediated cell death. Cell death of WT, IFNR −/− , ASC −/− , and caspase-1 (casp1) −/− BMMs was assayed by lactate dehydrogenase (LDH) release. BMMs either unactivated (B) or preactivated with heat-killed F. novicida (A, C, and D [left]) or pretreated with recombinant IFN-β (D, right) were infected for 8 (A), 12.5 (B), 3 (C), or 6 h (D) with F. novicida (A, B, and D) or S. typhimurium (C) strains at the indicated MOI. In agreement with previous data ( 30 ), cell death required the S. typhimurium gene sipB . Error bars represent SEM.
    Figure Legend Snippet: Type I IFN induction and signaling is required for F. novicida –mediated but not for S. typhimurium –mediated cell death. Cell death of WT, IFNR −/− , ASC −/− , and caspase-1 (casp1) −/− BMMs was assayed by lactate dehydrogenase (LDH) release. BMMs either unactivated (B) or preactivated with heat-killed F. novicida (A, C, and D [left]) or pretreated with recombinant IFN-β (D, right) were infected for 8 (A), 12.5 (B), 3 (C), or 6 h (D) with F. novicida (A, B, and D) or S. typhimurium (C) strains at the indicated MOI. In agreement with previous data ( 30 ), cell death required the S. typhimurium gene sipB . Error bars represent SEM.

    Techniques Used: Recombinant, Infection

    F. novicida in the host cytosol induces IFN-β secretion in a TLR-independent IRF-3–dependent manner. IFN-β mRNA levels were determined by quantitative RT-PCR in uninfected BMMs (Un) or at various times PI (A) and 7 (C), 9 (D), or 8 (E) h PI with either mglA or WT F. novicida . IFN-β levels were determined by ELISA in the supernatant of BMMs infected at the indicated MOI for 9 h (ND, nondetectable; B). Various vacuole-restricted mutants do not induce IFN-β mRNA, whereas their complemented counterparts (c.) do. Cytosolic localization is shown (C). BMMs from WT or from IRF-3 −/− , ASC −/− , MyD88/TRIF DKO , Ipaf −/− , RIP2 −/− (D), MAVS −/− mice, or WT littermates (E) were analyzed for their IFN-β mRNA levels. Error bars represent SEM.
    Figure Legend Snippet: F. novicida in the host cytosol induces IFN-β secretion in a TLR-independent IRF-3–dependent manner. IFN-β mRNA levels were determined by quantitative RT-PCR in uninfected BMMs (Un) or at various times PI (A) and 7 (C), 9 (D), or 8 (E) h PI with either mglA or WT F. novicida . IFN-β levels were determined by ELISA in the supernatant of BMMs infected at the indicated MOI for 9 h (ND, nondetectable; B). Various vacuole-restricted mutants do not induce IFN-β mRNA, whereas their complemented counterparts (c.) do. Cytosolic localization is shown (C). BMMs from WT or from IRF-3 −/− , ASC −/− , MyD88/TRIF DKO , Ipaf −/− , RIP2 −/− (D), MAVS −/− mice, or WT littermates (E) were analyzed for their IFN-β mRNA levels. Error bars represent SEM.

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, Mouse Assay

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    Incubation:

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation
    Article Snippet: .. The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ). .. After 3 hours incubation at room temperature, unbound materials were removed and the beads were washed in RIPA buffer containing 2 mM DTT, 28 μM E-64, 1 mM PMSF and 5 μg/mL leupeptin and 10 U RNasin Plus per ml.

    Isolation:

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Lysis:

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation
    Article Snippet: .. The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ). .. After 3 hours incubation at room temperature, unbound materials were removed and the beads were washed in RIPA buffer containing 2 mM DTT, 28 μM E-64, 1 mM PMSF and 5 μg/mL leupeptin and 10 U RNasin Plus per ml.

    Article Title: The Cdk8/19-cyclin C transcription regulator functions in genome replication through metazoan Sld7
    Article Snippet: .. IP For coimmunoprecipitations of tagged Treslin/TICRR and MTBP from 293T cells, a 10-cm plate of transiently transfected 293T cells were lysed in 5× pellet volume of lysis buffer (20 mM HEPES, 150 mM NaCl, 10% glycerol, complete EDTA-free protease inhibitor cocktail [Roche, 05056489001], 0.1% Triton X-100, 2 mM 2-mercaptoethanol). .. For flag affinity purifications 50% of cell lysates was incubated with 1 μg anti-FLAG mouse monoclonal antibody or 1 μg mouse IgG coupled to 150 μg Protein G Dynabeads (Invitrogen, 100-04D).

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    Roche cathepsin b cell lysis buffer kit
    <t>Cathepsin</t> B inhibition by E64 does not lead to α-synuclein accumulation
    Cathepsin B Cell Lysis Buffer Kit, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    cathepsin b cell lysis buffer kit - by Bioz Stars, 2020-09
    85/100 stars
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    92
    Roche lysis buffer b
    <t>Cathepsin</t> B inhibition by E64 does not lead to α-synuclein accumulation
    Lysis Buffer B, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer b/product/Roche
    Average 92 stars, based on 53 article reviews
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    lysis buffer b - by Bioz Stars, 2020-09
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    85
    Roche ripa b lysis buffer
    <t>Cathepsin</t> B inhibition by E64 does not lead to α-synuclein accumulation
    Ripa B Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ripa b lysis buffer/product/Roche
    Average 85 stars, based on 1 article reviews
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    ripa b lysis buffer - by Bioz Stars, 2020-09
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    Cathepsin B inhibition by E64 does not lead to α-synuclein accumulation

    Journal: Journal of neurochemistry

    Article Title: Overexpression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells*

    doi: 10.1111/jnc.12497

    Figure Lengend Snippet: Cathepsin B inhibition by E64 does not lead to α-synuclein accumulation

    Article Snippet: Briefly, cells were collected by scraping and centrifugation (1500 x g for 5 minutes at 4°C) and then lysed using cathepsin B cell lysis buffer (kit) with added protease (Roche) and phosphatase (Sigma) inhibitors followed by 30 minutes incubation on ice.

    Techniques: Inhibition