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  • 99
    Bio X Cell rat igg2a isotype control
    Rat Igg2a Isotype Control, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 99/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mouse igg2a
    Mouse Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher igg2a
    Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson igg2a
    Expression of TIM-2 on CD4 T and B cells . (a) Expression of T cell immunoglobulin and mucin domain (TIM)-2 on activated CD4 T cells. Purified splenic CD4 T cells were stimulated by immobilized anti-CD3 monoclonal antibody (mAb) with or without anti-CD28 mAb and harvested at the indicated periods. Cells were stained with biotinylated RMT2-26 or control <t>IgG</t> followed by PE-labeled streptavidin. (b) Expression of TIM-2 on activated B cells. Purified splenic B cells were stimulated with the indicated combinations of anti-IgM Ab, anti-CD40 mAb, and IL-4. Cells were harvested at the indicated periods and stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with anti-TIM-2 mAb and the dotted lines indicate background staining with control IgG.
    Igg2a, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore igg2a
    Expression of TIM-2 on CD4 T and B cells . (a) Expression of T cell immunoglobulin and mucin domain (TIM)-2 on activated CD4 T cells. Purified splenic CD4 T cells were stimulated by immobilized anti-CD3 monoclonal antibody (mAb) with or without anti-CD28 mAb and harvested at the indicated periods. Cells were stained with biotinylated RMT2-26 or control <t>IgG</t> followed by PE-labeled streptavidin. (b) Expression of TIM-2 on activated B cells. Purified splenic B cells were stimulated with the indicated combinations of anti-IgM Ab, anti-CD40 mAb, and IL-4. Cells were harvested at the indicated periods and stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with anti-TIM-2 mAb and the dotted lines indicate background staining with control IgG.
    Igg2a, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen igg2a
    Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat <t>IgG</t> (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P
    Igg2a, supplied by Pharmingen, used in various techniques. Bioz Stars score: 94/100, based on 718 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad igg2a
    Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat <t>IgG</t> (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P
    Igg2a, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson rat igg2a
    MSP1-specific <t>IgG</t> antibody-secreting cells (ASC) appear transiently in peripheral blood during infection. The frequencies of ASC were determined by direct ELISpot assays as described in the experimental procedures. The numbers of MSP1-specific IgG ASC in peripheral blood, spleen and bone marrow following both a primary and secondary infection are presented as ASC per ml of blood, per spleen and per two femurs respectively. The values and error bars shown are the means and the standard errors of the mean (SEM) of 5 to 7 mice.
    Rat Igg2a, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    igg2a  (Abcam)
    99
    Abcam igg2a
    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human <t>IgG1</t> Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human <t>IgG</t> targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.
    Igg2a, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 443 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cedarlane anti mouse human mac 2 galectin 3 purified clone m3 38 rat igg2a
    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human <t>IgG1</t> Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human <t>IgG</t> targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.
    Anti Mouse Human Mac 2 Galectin 3 Purified Clone M3 38 Rat Igg2a, supplied by Cedarlane, used in various techniques. Bioz Stars score: 99/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher goat anti mouse igg2a cross adsorbed secondary antibody
    COX-2 interacts with IRE1α in vivo . ( A ) Immunoprecipitation ( IP ) of endogenous IRE1α from HEK293 cells with anti-IRE1α or anti-COX-2 -antibodies. Immunoblot ( IB ) analysis was carried out with anti-IRE1α or anti-COX-2 antibodies. The location of IRE1α and COX-2 are indicated by the arrows. Immunoprecipitation experiments were performed in triplicate with representative blots shown. ( B ) His-tagged ER luminal domain of IRE1α ( IRE1-NLD ) was expressed in COS-1 cells followed by immunoprecipitation with anti-COX-2, anti-His-tag antibodies or <t>IgG.</t> Immunoblot ( IB ) analysis was carried out with anti-His antibodies. The location of IRE1-NLD is indicated by the arrow. Immunoprecipitation experiments were performed in triplicate with representative blot shown. ( C ) Pull-down of COX-2 in COS-1 cells expressing His-tagged IRE1-NLD. Upper blot was probed with anti-COX-2 antibodies. The lower blot was probed with anti-His-tag antibodies. Protein samples were separated on the same SDS-PAGE, the middle empty lanes were removed from the lower blot for the sake of clarity. Pull-down assay was performed in triplicate. The images of ( A – C .
    Goat Anti Mouse Igg2a Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson mouse igg2a
    Involvement of clathrin-mediated endocytic internalization in nuclear translocation of cell membrane ErbB-2. (A) MCF-7/HER18 cells were biotinylated at 4°C using normal human serum-biotin. The biotinylated molecules on the cell surface were released to internalize by incubation at 37°C for the indicated times. Nuclear extracts were then affinity purified with avidin-conjugated agarose and analyzed by Western blotting with anti-ErbB-2 antibody. (B) Cell lysates from MCF-7/HER18 cells were immunoprecipitated (IP) with anti-ErbB-2, anti-importin β1, mouse <t>IgG</t> (mIgG), or rIgG. The precipitated immunocomplexes were then analyzed by Western blotting with adaptin, clathrin, importin β1, and ErbB-2. (C) Immunofluorescence staining of ErbB-2, importin β1, and the endocytosis proteins adaptin and EPS15 visualized them under confocal microscopy. Top and middle panels: endogenous adaptin (green) and ErbB-2 (red) (top panels) and adaptin (green) and importin β1 (red) (middle panels) colocalized in MCF-7/HER18 cells. Bottom panels: GFP-EPS15 expression vectors were cotransfected with ErbB-2 into CHO cells. The cells were then immunostained for ErbB-2 (red) and studied by confocal microscopy. Arrowheads in the insets indicate nuclear colocalization.
    Mouse Igg2a, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 645 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rat igg2a
    Involvement of clathrin-mediated endocytic internalization in nuclear translocation of cell membrane ErbB-2. (A) MCF-7/HER18 cells were biotinylated at 4°C using normal human serum-biotin. The biotinylated molecules on the cell surface were released to internalize by incubation at 37°C for the indicated times. Nuclear extracts were then affinity purified with avidin-conjugated agarose and analyzed by Western blotting with anti-ErbB-2 antibody. (B) Cell lysates from MCF-7/HER18 cells were immunoprecipitated (IP) with anti-ErbB-2, anti-importin β1, mouse <t>IgG</t> (mIgG), or rIgG. The precipitated immunocomplexes were then analyzed by Western blotting with adaptin, clathrin, importin β1, and ErbB-2. (C) Immunofluorescence staining of ErbB-2, importin β1, and the endocytosis proteins adaptin and EPS15 visualized them under confocal microscopy. Top and middle panels: endogenous adaptin (green) and ErbB-2 (red) (top panels) and adaptin (green) and importin β1 (red) (middle panels) colocalized in MCF-7/HER18 cells. Bottom panels: GFP-EPS15 expression vectors were cotransfected with ErbB-2 into CHO cells. The cells were then immunostained for ErbB-2 (red) and studied by confocal microscopy. Arrowheads in the insets indicate nuclear colocalization.
    Rat Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 627 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology igg2a
    Duration of serum FMDV-specific <t>IgG</t> responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological
    Igg2a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio X Cell rat igg2a
    Duration of serum FMDV-specific <t>IgG</t> responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological
    Rat Igg2a, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 99/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems rat igg2a isotype control
    Duration of serum FMDV-specific <t>IgG</t> responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological
    Rat Igg2a Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rat igg2a isotype control
    Duration of serum FMDV-specific <t>IgG</t> responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological
    Rat Igg2a Isotype Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse igg2a
    Intracellular interaction between βCTF and TMEM30A. ], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse <t>IgG</t> or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.
    Mouse Igg2a, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rat igg2a kappa isotype control
    Intracellular interaction between βCTF and TMEM30A. ], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse <t>IgG</t> or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.
    Rat Igg2a Kappa Isotype Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies igg2a
    Involvement of HAMA in FcγR-mediated trogocytosis. ( A ) Effects of HAMA blockade on FcγR-mediated trogocytosis (n = 10). Heparinized whole blood samples were added with or without 1 µl of HAMA inhibitor, TRU block, and then made to react with the PE-labeled anti-CD8α (HIT8a) and FITC-labeled anti-CD15 (H198) Abs. After depletion of erythrocytes, these cells were subjected to FCM. PE-labeled mouse <t>IgG1</t> and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. Student t -test for paired samples was applied for statistical analysis. ( B ) Difference in the inhibition rates of FcγR-mediated trogocytosis by the HAMA inhibitor between HAMA high sera and HAMA low sera. The serum samples were divided into 2 groups, including HAMA high serum (more than 10 ng/ml, n = 5) and HAMA low serum (less than 10 ng/ml, n = 5). The decrease rates of CD8 + granulocytes by treatment with TRU block were calculated from the data presented in Figure 6A . Student t -test for unpaired samples was applied for statistical analysis.
    Igg2a, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bethyl igg2a
    Control of renal inflammation by gut microbiota in female lpr mice. a Serum level of <t>IgG2a</t> ( n = 7 mice per group; *** P
    Igg2a, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems mouse igg2a isotype control
    TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes. (A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control <t>IgG,</t> at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2 -ΔΔCT , where ΔΔC T is the average difference between the siTACSTD2 ΔC T and siControl ΔC T .
    Mouse Igg2a Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend mouse igg1
    TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes. (A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control <t>IgG,</t> at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2 -ΔΔCT , where ΔΔC T is the average difference between the siTACSTD2 ΔC T and siControl ΔC T .
    Mouse Igg1, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems igg2a
    TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes. (A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control <t>IgG,</t> at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2 -ΔΔCT , where ΔΔC T is the average difference between the siTACSTD2 ΔC T and siControl ΔC T .
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    Image Search Results


    Expression of TIM-2 on CD4 T and B cells . (a) Expression of T cell immunoglobulin and mucin domain (TIM)-2 on activated CD4 T cells. Purified splenic CD4 T cells were stimulated by immobilized anti-CD3 monoclonal antibody (mAb) with or without anti-CD28 mAb and harvested at the indicated periods. Cells were stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. (b) Expression of TIM-2 on activated B cells. Purified splenic B cells were stimulated with the indicated combinations of anti-IgM Ab, anti-CD40 mAb, and IL-4. Cells were harvested at the indicated periods and stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with anti-TIM-2 mAb and the dotted lines indicate background staining with control IgG.

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Expression of TIM-2 on CD4 T and B cells . (a) Expression of T cell immunoglobulin and mucin domain (TIM)-2 on activated CD4 T cells. Purified splenic CD4 T cells were stimulated by immobilized anti-CD3 monoclonal antibody (mAb) with or without anti-CD28 mAb and harvested at the indicated periods. Cells were stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. (b) Expression of TIM-2 on activated B cells. Purified splenic B cells were stimulated with the indicated combinations of anti-IgM Ab, anti-CD40 mAb, and IL-4. Cells were harvested at the indicated periods and stained with biotinylated RMT2-26 or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with anti-TIM-2 mAb and the dotted lines indicate background staining with control IgG.

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Expressing, Purification, Staining, Labeling

    Effect of anti-TIM-2 mAbs on Ig production in vitro . Purified B cells from FcRγ-deficient mice were stimulated with anti-IgM, anti-CD40, and IL-4 in the presence of the indicated anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) or control IgG. Culture supernatants were harvested at day 7 and analyzed using a CBA assay that could identify all seven mouse Ig isotypes in a single sample. Presence of a particular isotype is indicated by lambda (upper) and kappa (lower) fluorescence (with labels provided to indicate positive cultures). Data are representative of three experiments.

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Effect of anti-TIM-2 mAbs on Ig production in vitro . Purified B cells from FcRγ-deficient mice were stimulated with anti-IgM, anti-CD40, and IL-4 in the presence of the indicated anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) or control IgG. Culture supernatants were harvested at day 7 and analyzed using a CBA assay that could identify all seven mouse Ig isotypes in a single sample. Presence of a particular isotype is indicated by lambda (upper) and kappa (lower) fluorescence (with labels provided to indicate positive cultures). Data are representative of three experiments.

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: In Vitro, Purification, Mouse Assay, Crocin Bleaching Assay, Fluorescence

    Effect of anti-TIM-2 mAbs at different phases of CIA . (a-b) Exacerbation of collagen-induced arthritis (CIA) by RMT2-14 treatment. DBA/1 mice were immunized with primary type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and secondary CII/incomplete Freund's adjuvant (IFA) on day 21. Two groups of mice were treated with RMT2-14 or control IgG every three days from day 0 to day 42. (a) Clinical score and (b) incidence of arthritis were evaluated from day 0. (c) Effect of anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) at the early phase of CIA. Mice were immunized with CII/CFA once on day 0 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 0 to day 17. Clinical score of arthritis was evaluated from day 0. (d) Effect of anti-TIM-2 mAbs at the late phase of CIA. Mice were immunized with CII/CFA once on day 0 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 15 to day 32. Clinical score of arthritis was evaluated from day 0. Results are presented as the mean ± standard error of the mean of 10 mice in each group. *, P

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Effect of anti-TIM-2 mAbs at different phases of CIA . (a-b) Exacerbation of collagen-induced arthritis (CIA) by RMT2-14 treatment. DBA/1 mice were immunized with primary type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and secondary CII/incomplete Freund's adjuvant (IFA) on day 21. Two groups of mice were treated with RMT2-14 or control IgG every three days from day 0 to day 42. (a) Clinical score and (b) incidence of arthritis were evaluated from day 0. (c) Effect of anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) at the early phase of CIA. Mice were immunized with CII/CFA once on day 0 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 0 to day 17. Clinical score of arthritis was evaluated from day 0. (d) Effect of anti-TIM-2 mAbs at the late phase of CIA. Mice were immunized with CII/CFA once on day 0 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 15 to day 32. Clinical score of arthritis was evaluated from day 0. Results are presented as the mean ± standard error of the mean of 10 mice in each group. *, P

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Mouse Assay, Immunofluorescence

    Effect of anti-TIM-2 mAbs on histopathological arthritis . Hind paws from normal mice and control IgG- or RMT2-14-treated collagen-induced arthritis (CIA) mice at day 45 were stained with H E. Original magnification, × 4. Representatives in each group of 10 mice are shown.

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Effect of anti-TIM-2 mAbs on histopathological arthritis . Hind paws from normal mice and control IgG- or RMT2-14-treated collagen-induced arthritis (CIA) mice at day 45 were stained with H E. Original magnification, × 4. Representatives in each group of 10 mice are shown.

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Mouse Assay, Staining

    Effect of anti-TIM-2 mAb treatment on antigen-specific T cell proliferation and cytokine production . (a) DBA/1 mice were immunized with type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and CII/incomplete Freund's adjuvant (IFA) on day 21 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 0 to day 42. Draining lymph node (LN) cells from 19 mice were isolated and pooled in each group at day 45 and cultured with the indicated concentrations of denatured CII (dCII). For estimating proliferation, 0.5 μCi 3 H-thymidine was added during the last eight hours of a 96-hour culture. Production of IFN-γ and IL-17 in the culture supernatants at 120 hours was determined by ELISA. (b) DBA/1 mice were immunized with ovalbumin (OVA)/CFA on day 0 and treated with RMT2-14 or control IgG on days 0, 2, and 4. Draining LN cells from five mice were isolated and pooled in each group on day 7 and cultured with the indicated concentrations of OVA. For estimating proliferation, 0.5 μCi 3 H-thymidine was added during the last six hours of a 72-hour culture. Production of IFN-γ and IL-17 in the culture supernatants at 72 hours was determined by ELISA. Results are expressed as the mean ± standard deviation of triplicate samples. Similar results were obtained in three independent experiments.

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Effect of anti-TIM-2 mAb treatment on antigen-specific T cell proliferation and cytokine production . (a) DBA/1 mice were immunized with type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and CII/incomplete Freund's adjuvant (IFA) on day 21 and treated with RMT2-14, RMT2-25, RMT2-26, or control IgG from day 0 to day 42. Draining lymph node (LN) cells from 19 mice were isolated and pooled in each group at day 45 and cultured with the indicated concentrations of denatured CII (dCII). For estimating proliferation, 0.5 μCi 3 H-thymidine was added during the last eight hours of a 96-hour culture. Production of IFN-γ and IL-17 in the culture supernatants at 120 hours was determined by ELISA. (b) DBA/1 mice were immunized with ovalbumin (OVA)/CFA on day 0 and treated with RMT2-14 or control IgG on days 0, 2, and 4. Draining LN cells from five mice were isolated and pooled in each group on day 7 and cultured with the indicated concentrations of OVA. For estimating proliferation, 0.5 μCi 3 H-thymidine was added during the last six hours of a 72-hour culture. Production of IFN-γ and IL-17 in the culture supernatants at 72 hours was determined by ELISA. Results are expressed as the mean ± standard deviation of triplicate samples. Similar results were obtained in three independent experiments.

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Mouse Assay, Immunofluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Effect of anti-TIM-2 mAb treatment on serum anti-CII IgG titers . (a) DBA/1 mice were immunized with type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and treated with RMT2-14 or control IgG from day 0 to day 17. Serum levels of anti-CII IgG1, IgG2a, and IgG2b were measured by ELISA on day 16 and 24 after immunization. (b) DBA/1 mice were immunized with CII/CFA on day 0 and treated with RMT2-25, RMT2-26, or control IgG from day 0 to day 17. Serum levels of anti-CII IgG1, IgG2a, and IgG2b were measured by ELISA on day 32. Results are expressed as the mean ± standard error of the mean of 10 mice in each group. *, P

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Effect of anti-TIM-2 mAb treatment on serum anti-CII IgG titers . (a) DBA/1 mice were immunized with type II collagen (CII)/complete Freund's adjuvant (CFA) on day 0 and treated with RMT2-14 or control IgG from day 0 to day 17. Serum levels of anti-CII IgG1, IgG2a, and IgG2b were measured by ELISA on day 16 and 24 after immunization. (b) DBA/1 mice were immunized with CII/CFA on day 0 and treated with RMT2-25, RMT2-26, or control IgG from day 0 to day 17. Serum levels of anti-CII IgG1, IgG2a, and IgG2b were measured by ELISA on day 32. Results are expressed as the mean ± standard error of the mean of 10 mice in each group. *, P

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Characterization of anti-TIM-2 mAbs . (a) Reactivity of anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) to mouse TIM family molecules. NRK-52E-derived TIM transfectants and parental cells were stained with biotinylated RMT2-14, RMT2-25, RMT2-26, specific mAbs against each TIM family molecule, or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with the respective mAb and the dotted lines indicate background staining with control IgG. (b) H-ferritin binds to TIM-2 transfectant. NRK-52E-derived TIM transfectants were stained with Alexa647-labeled H-ferritin. Thick lines indicate the staining with the H-ferritin and the dotted lines indicate background staining with PBS. (c) Anti-TIM-2 mAbs inhibit H-ferritin binding to TIM-2 transfectant. TIM-2/L5178Y cells were pretreated with the indicated anti-TIM-2 mAb (thick lines) or control IgG (solid lines) and then stained with Alexa647-labeled H-ferritin. The dotted lines indicate background staining with PBS.

    Journal: Arthritis Research & Therapy

    Article Title: Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

    doi: 10.1186/ar3288

    Figure Lengend Snippet: Characterization of anti-TIM-2 mAbs . (a) Reactivity of anti-T cell immunoglobulin and mucin domain (TIM)-2 monoclonal antibodies (mAbs) to mouse TIM family molecules. NRK-52E-derived TIM transfectants and parental cells were stained with biotinylated RMT2-14, RMT2-25, RMT2-26, specific mAbs against each TIM family molecule, or control IgG followed by PE-labeled streptavidin. Thick lines indicate the staining with the respective mAb and the dotted lines indicate background staining with control IgG. (b) H-ferritin binds to TIM-2 transfectant. NRK-52E-derived TIM transfectants were stained with Alexa647-labeled H-ferritin. Thick lines indicate the staining with the H-ferritin and the dotted lines indicate background staining with PBS. (c) Anti-TIM-2 mAbs inhibit H-ferritin binding to TIM-2 transfectant. TIM-2/L5178Y cells were pretreated with the indicated anti-TIM-2 mAb (thick lines) or control IgG (solid lines) and then stained with Alexa647-labeled H-ferritin. The dotted lines indicate background staining with PBS.

    Article Snippet: After washing, biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b mAbs (BD Biosciences, San Jose, CA, USA) were added and incubated for one hour at 37°C, washed, and then developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and o-phenylendiamine (Wako Pure Chemical Industries, Osaka, Japan).

    Techniques: Derivative Assay, Staining, Labeling, Transfection, Binding Assay

    Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat IgG (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat IgG (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Mouse Assay

    Production of donor-reactive IgG antibody in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− kidney allograft recipients Serum was collected from individual wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients of A/J kidney allografts on days 3, 7, and 14 post-transplant and at the time of rejection. The sera were tested for reactivity to A/J thymocytes using a flow cytometry-based approach to determine the titer of donor-reactive antibody for each of the IgG subclasses. Data indicate mean titer for each recipient group ± SEM.

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Production of donor-reactive IgG antibody in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− kidney allograft recipients Serum was collected from individual wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients of A/J kidney allografts on days 3, 7, and 14 post-transplant and at the time of rejection. The sera were tested for reactivity to A/J thymocytes using a flow cytometry-based approach to determine the titer of donor-reactive antibody for each of the IgG subclasses. Data indicate mean titer for each recipient group ± SEM.

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Flow Cytometry, Cytometry

    Titers of donor-reactive IgG antibody at the time of kidney allograft rejection in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients (a) Titers of donor-reactive IgG antibody were determined in serum collected from allograft recipients at the time of graft rejection or from C57BL/7 recipients with long-term surviving allografts collected on day 40 post-transplant. Data indicate individual titers within each recipient group and the mean titer for each recipient group ± SEM. (b–d) Histological evaluation of a rejecting kidney allograft from a wild C57BL/6 recipient on day 36 post-transplant stained with: (b) hematoxylin and eosin; (c) anti-C4d antibody; and (d) anti-Mac2 antibody. Arrows indicate marginating leukocytes. Images shown are representative of 2 individual allografts in the group.

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Titers of donor-reactive IgG antibody at the time of kidney allograft rejection in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients (a) Titers of donor-reactive IgG antibody were determined in serum collected from allograft recipients at the time of graft rejection or from C57BL/7 recipients with long-term surviving allografts collected on day 40 post-transplant. Data indicate individual titers within each recipient group and the mean titer for each recipient group ± SEM. (b–d) Histological evaluation of a rejecting kidney allograft from a wild C57BL/6 recipient on day 36 post-transplant stained with: (b) hematoxylin and eosin; (c) anti-C4d antibody; and (d) anti-Mac2 antibody. Arrows indicate marginating leukocytes. Images shown are representative of 2 individual allografts in the group.

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Staining

    Expression of inflammatory mediator mRNA in kidney allografts in B6.CD8 −/− CCR5 −/− recipients treated with mAb depleting NK or Gr-1 + cells Wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− received renal allografts from A/J donors and a group of wild type C57BL/6 mice received isografts. Groups of 3–5 recipients were treated with 250 µg anti-Gr-1 (RB6.8C5) or anti-NK 1.1 (PK136) mAb to deplete Gr-1 + or NK cells or rat IgG as a control on days 5 and 6 post-transplant. Grafts were harvested on day 7 post-transplant and whole cell RNA was prepared and analyzed by qRT/PCR for expression of the indicated cytokine, chemokine and inflammatory mediator genes. Data indicate the mean expression levels of each test gene in allografts for each group. * P

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Expression of inflammatory mediator mRNA in kidney allografts in B6.CD8 −/− CCR5 −/− recipients treated with mAb depleting NK or Gr-1 + cells Wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− received renal allografts from A/J donors and a group of wild type C57BL/6 mice received isografts. Groups of 3–5 recipients were treated with 250 µg anti-Gr-1 (RB6.8C5) or anti-NK 1.1 (PK136) mAb to deplete Gr-1 + or NK cells or rat IgG as a control on days 5 and 6 post-transplant. Grafts were harvested on day 7 post-transplant and whole cell RNA was prepared and analyzed by qRT/PCR for expression of the indicated cytokine, chemokine and inflammatory mediator genes. Data indicate the mean expression levels of each test gene in allografts for each group. * P

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    MSP1-specific IgG antibody-secreting cells (ASC) appear transiently in peripheral blood during infection. The frequencies of ASC were determined by direct ELISpot assays as described in the experimental procedures. The numbers of MSP1-specific IgG ASC in peripheral blood, spleen and bone marrow following both a primary and secondary infection are presented as ASC per ml of blood, per spleen and per two femurs respectively. The values and error bars shown are the means and the standard errors of the mean (SEM) of 5 to 7 mice.

    Journal: PLoS ONE

    Article Title: Distinct Kinetics of Memory B-Cell and Plasma-Cell Responses in Peripheral Blood Following a Blood-Stage Plasmodium chabaudi Infection in Mice

    doi: 10.1371/journal.pone.0015007

    Figure Lengend Snippet: MSP1-specific IgG antibody-secreting cells (ASC) appear transiently in peripheral blood during infection. The frequencies of ASC were determined by direct ELISpot assays as described in the experimental procedures. The numbers of MSP1-specific IgG ASC in peripheral blood, spleen and bone marrow following both a primary and secondary infection are presented as ASC per ml of blood, per spleen and per two femurs respectively. The values and error bars shown are the means and the standard errors of the mean (SEM) of 5 to 7 mice.

    Article Snippet: Anti-Fc receptor and Rat IgG2b'κ FITC, Rat IgG2a'κ PE, Rat IgG2a'κ biotin, Rat (lavian) IgMκ isotype controls were purchased from BD Pharmingen.

    Techniques: Infection, Enzyme-linked Immunospot, Mouse Assay

    MSP1-specific IgG memory B cells are detectable in peripheral blood in low numbers. The frequencies of MSP1-specific IgG memory B cells were determined by in vitro cultured ELISpot assays as described in the experimental procedures. The frequencies of MSP1-specific IgG memory B cells in peripheral blood and spleen following both a primary and secondary infection are presented as memory B cells per ml of blood and per spleen respectively. The values and error bars shown are the means and the standard errors of the mean (SEM) of 5 to 7 mice.

    Journal: PLoS ONE

    Article Title: Distinct Kinetics of Memory B-Cell and Plasma-Cell Responses in Peripheral Blood Following a Blood-Stage Plasmodium chabaudi Infection in Mice

    doi: 10.1371/journal.pone.0015007

    Figure Lengend Snippet: MSP1-specific IgG memory B cells are detectable in peripheral blood in low numbers. The frequencies of MSP1-specific IgG memory B cells were determined by in vitro cultured ELISpot assays as described in the experimental procedures. The frequencies of MSP1-specific IgG memory B cells in peripheral blood and spleen following both a primary and secondary infection are presented as memory B cells per ml of blood and per spleen respectively. The values and error bars shown are the means and the standard errors of the mean (SEM) of 5 to 7 mice.

    Article Snippet: Anti-Fc receptor and Rat IgG2b'κ FITC, Rat IgG2a'κ PE, Rat IgG2a'κ biotin, Rat (lavian) IgMκ isotype controls were purchased from BD Pharmingen.

    Techniques: In Vitro, Cell Culture, Enzyme-linked Immunospot, Infection, Mouse Assay

    Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human IgG1 Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human IgG targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.

    Journal: PLoS Pathogens

    Article Title: Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B

    doi: 10.1371/journal.ppat.1008609

    Figure Lengend Snippet: Expression and purification of the recombinant gH/gL/gQ1/gQ2 complex. (A) The constructions of gH, gL, gQ1, and gQ2 are shown. gH is modified to have an N-terminal IL-2 signal sequence and a C-terminal human IgG1 Fc (hFc) and His 6 sequence replacing the intrinsic gH signal sequence and transmembrane-cytoplasmic tail domains, respectively. ( B) Cell binding assay was performed using hCD134 expressing JJhan cells or JJhan cells (negative control). After incubation with the tetramer-hFc containing medium, the interaction between cell expressing hCD134 and tetramer-hFc was detected using Alexa 488 conjugated anti-human IgG targeting the hFc by flow cytometry. The hFc protein (without tetramer) was used as the non-binding negative control. ( C) Size exclusion column chromatography of the purified tetramer. ( D) Western blotting analysis of the tetramer purified by the size exclusion column chromatography. gH, gQ1, gL, and gQ2 were detected by the respective antibodies. ( E) Purified gH/gL/gQ1/gQ2 complex detected by Coomassie brilliant blue staining.

    Article Snippet: For the measurement of IgG1, IgG2a and IgG3, the first antibodies were diluted to 1:2560 and the secondary antibodies were added to HRP-conjugated anti-mouse IgG1, IgG2a or IgG3 (Abcam, Cambridge, UK).

    Techniques: Expressing, Purification, Recombinant, Modification, Sequencing, Cell Binding Assay, Negative Control, Incubation, Flow Cytometry, Binding Assay, Column Chromatography, Western Blot, Staining

    Humoral immunity induced by immunization with the tetramer and the combination of Alum and CpG adjuvant. (A) Immunization schedule indicated as the weeks of immunization of four-week-old female BALB/c mice (n = 5). ( B) The amounts of the anti-tetramer antibodies in the sera collected from mice immunized with the indicated combination of adjuvants at the eight weeks after the third immunization were evaluated for the further ELISA assay using purified tetramer. The titers were calculated as the maximum dilution at which the value of OD405 was higher than that of the mean+2SD of the group immunized with PBS. ( C) The IgG isotypes of the anti-tetramer antibodies were analyzed by the ELISA assay at the dilution of 2560. IgG1, 1gG2a and IgG3 were detected separately from the same sera. The values of serum estimated IgG concentration were obtained from standard curves for the isotype control antibodies of mouse IgG1, IgG2a and IgG3, and presented as the mean±SD. (D) The neutralizing activities of the sera from the immunized mice indicated were measured in MT4 cells. Titers are presented as the maximum dilution at which the HHV-6B IE1 protein could not be detected. Data are the mean±SD of each group. *p

    Journal: PLoS Pathogens

    Article Title: Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B

    doi: 10.1371/journal.ppat.1008609

    Figure Lengend Snippet: Humoral immunity induced by immunization with the tetramer and the combination of Alum and CpG adjuvant. (A) Immunization schedule indicated as the weeks of immunization of four-week-old female BALB/c mice (n = 5). ( B) The amounts of the anti-tetramer antibodies in the sera collected from mice immunized with the indicated combination of adjuvants at the eight weeks after the third immunization were evaluated for the further ELISA assay using purified tetramer. The titers were calculated as the maximum dilution at which the value of OD405 was higher than that of the mean+2SD of the group immunized with PBS. ( C) The IgG isotypes of the anti-tetramer antibodies were analyzed by the ELISA assay at the dilution of 2560. IgG1, 1gG2a and IgG3 were detected separately from the same sera. The values of serum estimated IgG concentration were obtained from standard curves for the isotype control antibodies of mouse IgG1, IgG2a and IgG3, and presented as the mean±SD. (D) The neutralizing activities of the sera from the immunized mice indicated were measured in MT4 cells. Titers are presented as the maximum dilution at which the HHV-6B IE1 protein could not be detected. Data are the mean±SD of each group. *p

    Article Snippet: For the measurement of IgG1, IgG2a and IgG3, the first antibodies were diluted to 1:2560 and the secondary antibodies were added to HRP-conjugated anti-mouse IgG1, IgG2a or IgG3 (Abcam, Cambridge, UK).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay

    COX-2 interacts with IRE1α in vivo . ( A ) Immunoprecipitation ( IP ) of endogenous IRE1α from HEK293 cells with anti-IRE1α or anti-COX-2 -antibodies. Immunoblot ( IB ) analysis was carried out with anti-IRE1α or anti-COX-2 antibodies. The location of IRE1α and COX-2 are indicated by the arrows. Immunoprecipitation experiments were performed in triplicate with representative blots shown. ( B ) His-tagged ER luminal domain of IRE1α ( IRE1-NLD ) was expressed in COS-1 cells followed by immunoprecipitation with anti-COX-2, anti-His-tag antibodies or IgG. Immunoblot ( IB ) analysis was carried out with anti-His antibodies. The location of IRE1-NLD is indicated by the arrow. Immunoprecipitation experiments were performed in triplicate with representative blot shown. ( C ) Pull-down of COX-2 in COS-1 cells expressing His-tagged IRE1-NLD. Upper blot was probed with anti-COX-2 antibodies. The lower blot was probed with anti-His-tag antibodies. Protein samples were separated on the same SDS-PAGE, the middle empty lanes were removed from the lower blot for the sake of clarity. Pull-down assay was performed in triplicate. The images of ( A – C .

    Journal: Scientific Reports

    Article Title: Cyclosporine A binding to COX-2 reveals a novel signaling pathway that activates the IRE1α unfolded protein response sensor

    doi: 10.1038/s41598-018-34891-w

    Figure Lengend Snippet: COX-2 interacts with IRE1α in vivo . ( A ) Immunoprecipitation ( IP ) of endogenous IRE1α from HEK293 cells with anti-IRE1α or anti-COX-2 -antibodies. Immunoblot ( IB ) analysis was carried out with anti-IRE1α or anti-COX-2 antibodies. The location of IRE1α and COX-2 are indicated by the arrows. Immunoprecipitation experiments were performed in triplicate with representative blots shown. ( B ) His-tagged ER luminal domain of IRE1α ( IRE1-NLD ) was expressed in COS-1 cells followed by immunoprecipitation with anti-COX-2, anti-His-tag antibodies or IgG. Immunoblot ( IB ) analysis was carried out with anti-His antibodies. The location of IRE1-NLD is indicated by the arrow. Immunoprecipitation experiments were performed in triplicate with representative blot shown. ( C ) Pull-down of COX-2 in COS-1 cells expressing His-tagged IRE1-NLD. Upper blot was probed with anti-COX-2 antibodies. The lower blot was probed with anti-His-tag antibodies. Protein samples were separated on the same SDS-PAGE, the middle empty lanes were removed from the lower blot for the sake of clarity. Pull-down assay was performed in triplicate. The images of ( A – C .

    Article Snippet: Cells were washed and incubated with goat ant-mouse (Alexa Fluor 594; life technologies; catalog # A21135) at dilution 1:50 or goat ant-rabbit (Alexa Fluor 488; life technologies; catalog # A11034) at dilution 1:50.

    Techniques: In Vivo, Immunoprecipitation, Expressing, SDS Page, Pull Down Assay

    Involvement of clathrin-mediated endocytic internalization in nuclear translocation of cell membrane ErbB-2. (A) MCF-7/HER18 cells were biotinylated at 4°C using normal human serum-biotin. The biotinylated molecules on the cell surface were released to internalize by incubation at 37°C for the indicated times. Nuclear extracts were then affinity purified with avidin-conjugated agarose and analyzed by Western blotting with anti-ErbB-2 antibody. (B) Cell lysates from MCF-7/HER18 cells were immunoprecipitated (IP) with anti-ErbB-2, anti-importin β1, mouse IgG (mIgG), or rIgG. The precipitated immunocomplexes were then analyzed by Western blotting with adaptin, clathrin, importin β1, and ErbB-2. (C) Immunofluorescence staining of ErbB-2, importin β1, and the endocytosis proteins adaptin and EPS15 visualized them under confocal microscopy. Top and middle panels: endogenous adaptin (green) and ErbB-2 (red) (top panels) and adaptin (green) and importin β1 (red) (middle panels) colocalized in MCF-7/HER18 cells. Bottom panels: GFP-EPS15 expression vectors were cotransfected with ErbB-2 into CHO cells. The cells were then immunostained for ErbB-2 (red) and studied by confocal microscopy. Arrowheads in the insets indicate nuclear colocalization.

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: Involvement of clathrin-mediated endocytic internalization in nuclear translocation of cell membrane ErbB-2. (A) MCF-7/HER18 cells were biotinylated at 4°C using normal human serum-biotin. The biotinylated molecules on the cell surface were released to internalize by incubation at 37°C for the indicated times. Nuclear extracts were then affinity purified with avidin-conjugated agarose and analyzed by Western blotting with anti-ErbB-2 antibody. (B) Cell lysates from MCF-7/HER18 cells were immunoprecipitated (IP) with anti-ErbB-2, anti-importin β1, mouse IgG (mIgG), or rIgG. The precipitated immunocomplexes were then analyzed by Western blotting with adaptin, clathrin, importin β1, and ErbB-2. (C) Immunofluorescence staining of ErbB-2, importin β1, and the endocytosis proteins adaptin and EPS15 visualized them under confocal microscopy. Top and middle panels: endogenous adaptin (green) and ErbB-2 (red) (top panels) and adaptin (green) and importin β1 (red) (middle panels) colocalized in MCF-7/HER18 cells. Bottom panels: GFP-EPS15 expression vectors were cotransfected with ErbB-2 into CHO cells. The cells were then immunostained for ErbB-2 (red) and studied by confocal microscopy. Arrowheads in the insets indicate nuclear colocalization.

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Translocation Assay, Incubation, Affinity Purification, Avidin-Biotin Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Confocal Microscopy, Expressing

    Inactivation of nuclear export receptor leads to accumulation of ErbB-2 in the nucleus. (A) Nuclear extracts from MCF-7/HER18 cells treated with 20-ng/ml LMB for the indicated times were analyzed for nuclear ErbB-2 by Western blot analysis using anti-ErbB-2 antibody. Similarly, aliquots of the nuclear extracts were also analyzed by Western blot analysis using indicated antibodies as positive controls. Absence of α-tubulin in the nucleus indicates that there is no cytoplasmic contamination in the nuclear extract. (B) MCF-7/HER18 cells were treated with 20-ng/ml LMB for 12 h and stained for ErbB-2 by immunofluorescence analysis and examined under a confocal laser microscope. ErbB-2 localization in the nucleus was recognized as white granules (arrowheads). (C) Immunogold staining of ultrathin sections for ErbB-2 showed the accumulation of ErbB-2 in the nucleus of control (left panel, inset) and in the nucleus of LMB-treated MCF-7/HER18 cells (right panel, inset). The gold particles labeled ErbB-2 (15 nm). Cy, cytoplasm; Nu, nucleus. (D) Nuclear lysates from MCF-7/HER18 cells were immunoprecipitated (IP) using mouse anti-ErbB-2 antibody or mouse IgG (mIgG) antibody and Western blotted with anti-Crm1 antibody (upper panel). Similarly, equal amounts of lysate were also used for reciprocal immunoprecipitation using rabbit anti-Crm1 antibody or rIgG antibody and blotted with anti-ErbB-2 antibody (lower panel).

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: Inactivation of nuclear export receptor leads to accumulation of ErbB-2 in the nucleus. (A) Nuclear extracts from MCF-7/HER18 cells treated with 20-ng/ml LMB for the indicated times were analyzed for nuclear ErbB-2 by Western blot analysis using anti-ErbB-2 antibody. Similarly, aliquots of the nuclear extracts were also analyzed by Western blot analysis using indicated antibodies as positive controls. Absence of α-tubulin in the nucleus indicates that there is no cytoplasmic contamination in the nuclear extract. (B) MCF-7/HER18 cells were treated with 20-ng/ml LMB for 12 h and stained for ErbB-2 by immunofluorescence analysis and examined under a confocal laser microscope. ErbB-2 localization in the nucleus was recognized as white granules (arrowheads). (C) Immunogold staining of ultrathin sections for ErbB-2 showed the accumulation of ErbB-2 in the nucleus of control (left panel, inset) and in the nucleus of LMB-treated MCF-7/HER18 cells (right panel, inset). The gold particles labeled ErbB-2 (15 nm). Cy, cytoplasm; Nu, nucleus. (D) Nuclear lysates from MCF-7/HER18 cells were immunoprecipitated (IP) using mouse anti-ErbB-2 antibody or mouse IgG (mIgG) antibody and Western blotted with anti-Crm1 antibody (upper panel). Similarly, equal amounts of lysate were also used for reciprocal immunoprecipitation using rabbit anti-Crm1 antibody or rIgG antibody and blotted with anti-ErbB-2 antibody (lower panel).

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Western Blot, Staining, Immunofluorescence, Microscopy, Labeling, Immunoprecipitation

    ErbB-2 mutant with deletion of nuclear localization signal is deficient in nuclear transport. (A) 293 cells were transfected with GFP-tagged wild-type ErbB-2 (WT), GFP-tagged ErbB-2 mutant containing a deficient nuclear localization signal (ΔNLS), or vector control (−). Proteins from the cytoplasmic fraction, nuclear fraction, and total cell lysates were then analyzed by Western blotting with antibodies as indicated. (B) MCF-7 cells were transfected with either GFP-tagged WT or GFP-tagged ΔNLS mutant ErbB-2, and the localization of the ErbB-2 proteins was shown by confocal microscopy. ErbB-2 expression in the nucleus was recognized as white granules (arrows). (C) Lysates from CHO cells transfected with either WT or mutant ΔNLS ErbB-2 constructs were immunoprecipitated (IP) using anti-ErbB-2 or mouse IgG (mIgG) antibodies, and the amount of associated importin β1 was determined by immunoblotting analysis. (D) 293 cells were transfected with the GFP-tagged WT ErbB-2, the GFP-tagged ΔNLS ErbB-2 mutant, or vector control and analyzed by fluorescence microscopy. (E) The GFP-tagged WT or ΔNLS ErbB-2 mutant or vector control (Vec) was cotransfected into cells with the Elk1 fusion transactivator plasmid and luciferase reporter plasmid. After 24 h, the cells were treated with (+) or without (−) tyrosine kinase inhibitor AG825 (80 μM) for 6 h. The luciferase activity induced by the vector alone without AG825 treatment was set as 100%, and the relative luciferase activities are presented as the means with standard deviations of three independent experiments performed in triplicate. Expression of ErbB-2, phosphorylated ErbB-2 (p-ErbB-2), total extracellular signal- regulated kinase (ERK), phosphorylated ERK (p-ERK), and α-tubulin proteins was analyzed by Western blotting. (F) SKBr3 cells were transfected with the GFP-tagged WT ErbB-2 or the GFP-tagged ΔNLS ErbB-2 mutant. Twenty-four hours after transfection, the cells were treated with (+) or without (−) heregulin (HRG) (6 ng/ml) for 4 days and analyzed by fluorescence microscopy. The bar diagram indicates the increase of ErbB-2 (GFP) fluorescence in the cytoplasm after cells were treated with heregulin, calculated for a total of 100 cells. The cytoplasm-positive cells were defined as ErbB-2 (GFP) negative in the cell membrane but positive in the cytoplasm. As long as ErbB-2 was detected in a cell membrane, the cell was considered membrane positive regardless of the cytoplasmic status of ErbB-2.

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: ErbB-2 mutant with deletion of nuclear localization signal is deficient in nuclear transport. (A) 293 cells were transfected with GFP-tagged wild-type ErbB-2 (WT), GFP-tagged ErbB-2 mutant containing a deficient nuclear localization signal (ΔNLS), or vector control (−). Proteins from the cytoplasmic fraction, nuclear fraction, and total cell lysates were then analyzed by Western blotting with antibodies as indicated. (B) MCF-7 cells were transfected with either GFP-tagged WT or GFP-tagged ΔNLS mutant ErbB-2, and the localization of the ErbB-2 proteins was shown by confocal microscopy. ErbB-2 expression in the nucleus was recognized as white granules (arrows). (C) Lysates from CHO cells transfected with either WT or mutant ΔNLS ErbB-2 constructs were immunoprecipitated (IP) using anti-ErbB-2 or mouse IgG (mIgG) antibodies, and the amount of associated importin β1 was determined by immunoblotting analysis. (D) 293 cells were transfected with the GFP-tagged WT ErbB-2, the GFP-tagged ΔNLS ErbB-2 mutant, or vector control and analyzed by fluorescence microscopy. (E) The GFP-tagged WT or ΔNLS ErbB-2 mutant or vector control (Vec) was cotransfected into cells with the Elk1 fusion transactivator plasmid and luciferase reporter plasmid. After 24 h, the cells were treated with (+) or without (−) tyrosine kinase inhibitor AG825 (80 μM) for 6 h. The luciferase activity induced by the vector alone without AG825 treatment was set as 100%, and the relative luciferase activities are presented as the means with standard deviations of three independent experiments performed in triplicate. Expression of ErbB-2, phosphorylated ErbB-2 (p-ErbB-2), total extracellular signal- regulated kinase (ERK), phosphorylated ERK (p-ERK), and α-tubulin proteins was analyzed by Western blotting. (F) SKBr3 cells were transfected with the GFP-tagged WT ErbB-2 or the GFP-tagged ΔNLS ErbB-2 mutant. Twenty-four hours after transfection, the cells were treated with (+) or without (−) heregulin (HRG) (6 ng/ml) for 4 days and analyzed by fluorescence microscopy. The bar diagram indicates the increase of ErbB-2 (GFP) fluorescence in the cytoplasm after cells were treated with heregulin, calculated for a total of 100 cells. The cytoplasm-positive cells were defined as ErbB-2 (GFP) negative in the cell membrane but positive in the cytoplasm. As long as ErbB-2 was detected in a cell membrane, the cell was considered membrane positive regardless of the cytoplasmic status of ErbB-2.

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Confocal Microscopy, Expressing, Construct, Immunoprecipitation, Fluorescence, Microscopy, Luciferase, Activity Assay

    ErbB-2 nuclear translocation requires endocytosis. (A) Dynamin activity is required for nuclear localization of ErbB-2. CHO cells were transfected with ErbB-2 cDNA together with GFP-tagged WT or GFP-tagged K44A mutant (K44A) dynamin 2 cDNA. Left panels: the nuclear lysates from these cells were analyzed by Western blotting using antibodies against ErbB-2, PARP, and α-tubulin. The levels of ErbB-2 expression were quantitated and normalized to the protein levels of PARP. The expression level of ErbB-2 in GFP-tagged WT dynamin 2-transfected cells was set as 1. Right panels: total cell lysates were blotted for equal expression of ErbB-2, α-tubulin, and dynamin 2 (GFP). (B) CHO cells were transfected with the GFP-tagged WT (top) or K44A mutant (bottom) dynamin 2 together with ErbB-2 plasmid. Localization of ErbB-2 (red) in the TOPRO 3-stained nucleus (blue) was examined under a confocal microscope. ErbB-2 localized in the nucleus is shown in pink spots as indicated by arrows in the inset. (C) Cytoplasmic fractions (C), nuclear fractions (N), and total cell lysates from MCF-7/WT ErbB-2, MCF-7/kinase-deficient mutant ErbB-2 (kd ErbB-2), or vector control cells were analyzed by Western blotting with antibodies as indicated. (D) Lysates from MCF-7/WT ErbB-2 and MCF-7/kd ErbB-2 cells were immunoprecipitated (IP) with ErbB-2, importin β1 (Imp), EEA1, control mouse IgG (mIgG), and rIgG. The immunocomplexes were then analyzed by Western blotting with antibodies as indicated. Total cell lysates were also analyzed by Western blotting with anti-ErbB-2, antiphosphotyrosine (p-ErbB-2), anti-importin β1, and anti-EEA1 antibodies as positive controls. The blots marked with asterisks were exposed for a longer period.

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: ErbB-2 nuclear translocation requires endocytosis. (A) Dynamin activity is required for nuclear localization of ErbB-2. CHO cells were transfected with ErbB-2 cDNA together with GFP-tagged WT or GFP-tagged K44A mutant (K44A) dynamin 2 cDNA. Left panels: the nuclear lysates from these cells were analyzed by Western blotting using antibodies against ErbB-2, PARP, and α-tubulin. The levels of ErbB-2 expression were quantitated and normalized to the protein levels of PARP. The expression level of ErbB-2 in GFP-tagged WT dynamin 2-transfected cells was set as 1. Right panels: total cell lysates were blotted for equal expression of ErbB-2, α-tubulin, and dynamin 2 (GFP). (B) CHO cells were transfected with the GFP-tagged WT (top) or K44A mutant (bottom) dynamin 2 together with ErbB-2 plasmid. Localization of ErbB-2 (red) in the TOPRO 3-stained nucleus (blue) was examined under a confocal microscope. ErbB-2 localized in the nucleus is shown in pink spots as indicated by arrows in the inset. (C) Cytoplasmic fractions (C), nuclear fractions (N), and total cell lysates from MCF-7/WT ErbB-2, MCF-7/kinase-deficient mutant ErbB-2 (kd ErbB-2), or vector control cells were analyzed by Western blotting with antibodies as indicated. (D) Lysates from MCF-7/WT ErbB-2 and MCF-7/kd ErbB-2 cells were immunoprecipitated (IP) with ErbB-2, importin β1 (Imp), EEA1, control mouse IgG (mIgG), and rIgG. The immunocomplexes were then analyzed by Western blotting with antibodies as indicated. Total cell lysates were also analyzed by Western blotting with anti-ErbB-2, antiphosphotyrosine (p-ErbB-2), anti-importin β1, and anti-EEA1 antibodies as positive controls. The blots marked with asterisks were exposed for a longer period.

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Translocation Assay, Activity Assay, Transfection, Mutagenesis, Western Blot, Expressing, Plasmid Preparation, Staining, Microscopy, Immunoprecipitation

    ErbB-2 interacts with importin β1. (A) Lysates of the cytoplasmic fraction from MCF-7, MCF-7/HER18, and MDA-MB-453 cells were immunoprecipitated (IP) with antibodies against ErbB-2, importin β1, control mouse IgG (mIgG), and rIgG. The presence of importin β1 and ErbB-2 in the immunocomplexes was examined by immunoblotting analysis. Total cell lysate from the MCF-7/HER18 cells was used as the positive control. (B) Nuclear lysates from the same cell lines were tested for the association between ErbB-2 and importin β1 as described for panel A. Total cell lysate from the MCF-7/HER18 cells was used as the positive control. (C) ErbB-2 and importin β1 colocalized in the cytoplasm (insets 1 and 2, arrowheads) and the nucleus (insets 1 and 2, arrows) of MCF-7/HER18 cells as shown by immunofluorescence staining using a mouse monoclonal anti-ErbB-2 antibody directed against the extracellular domain of ErbB-2 and a rabbit polyclonal anti-importin β1 antibody. The images were then analyzed by confocal microscopy. The boxed areas are shown in detail in insets 1 and 2. (D) Immunogold staining of ultrathin sections for ErbB-2 and importin β1 demonstrated their association in the cytoplasm (left, inset, arrowheads) and nucleus (right, inset, arrows) of MCF-7/HER18 cells. The large and small gold particles labeled ErbB-2 (15 nm) and importin β1 (5 nm), respectively. Bar = 200 nm. Cy, cytoplasm; Nu, nucleus; PM, plasma membrane. (E) MCF-7/HER18 cells were transfected with importin β1 siRNA (Imp), nonspecific siRNA control (N.S.), or buffer only (−). Proteins from the cytoplasmic, nuclear, and total cell lysates were then analyzed by Western blotting with antibodies as indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: ErbB-2 interacts with importin β1. (A) Lysates of the cytoplasmic fraction from MCF-7, MCF-7/HER18, and MDA-MB-453 cells were immunoprecipitated (IP) with antibodies against ErbB-2, importin β1, control mouse IgG (mIgG), and rIgG. The presence of importin β1 and ErbB-2 in the immunocomplexes was examined by immunoblotting analysis. Total cell lysate from the MCF-7/HER18 cells was used as the positive control. (B) Nuclear lysates from the same cell lines were tested for the association between ErbB-2 and importin β1 as described for panel A. Total cell lysate from the MCF-7/HER18 cells was used as the positive control. (C) ErbB-2 and importin β1 colocalized in the cytoplasm (insets 1 and 2, arrowheads) and the nucleus (insets 1 and 2, arrows) of MCF-7/HER18 cells as shown by immunofluorescence staining using a mouse monoclonal anti-ErbB-2 antibody directed against the extracellular domain of ErbB-2 and a rabbit polyclonal anti-importin β1 antibody. The images were then analyzed by confocal microscopy. The boxed areas are shown in detail in insets 1 and 2. (D) Immunogold staining of ultrathin sections for ErbB-2 and importin β1 demonstrated their association in the cytoplasm (left, inset, arrowheads) and nucleus (right, inset, arrows) of MCF-7/HER18 cells. The large and small gold particles labeled ErbB-2 (15 nm) and importin β1 (5 nm), respectively. Bar = 200 nm. Cy, cytoplasm; Nu, nucleus; PM, plasma membrane. (E) MCF-7/HER18 cells were transfected with importin β1 siRNA (Imp), nonspecific siRNA control (N.S.), or buffer only (−). Proteins from the cytoplasmic, nuclear, and total cell lysates were then analyzed by Western blotting with antibodies as indicated.

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Multiple Displacement Amplification, Immunoprecipitation, Positive Control, Immunofluorescence, Staining, Confocal Microscopy, Labeling, Transfection, Western Blot

    Nuclear translocation of ErbB-2 by endosomal sorting. (A) Model for importin β1-mediated ErbB-2 nuclear transport by endosomal sorting. The scale of the diagram does not reflect the relative sizes of different molecules and the subcellular structures. (B) ErbB-2 and importin β1 colocalized in the endosome wall by electron microscopy. An ultrathin section of MCF-7/HER18 cells was immunostained for ErbB-2 (15-nm gold particles, thick arrow) and importin β1 (5-nm gold particles, thin arrow). The arrowhead indicates colocalization of the two proteins in the endosome (E) wall. (C) ErbB-2 and EEA1 colocalization in the cytoplasm and nucleus was demonstrated by electron microscopy. Ultrathin sections from MCF-7/HER18 cells were immunostained for ErbB-2 (5-nm gold particles) and EEA1 (15-nm gold particles). Inset 1 reveals the colocalization of ErbB-2 and EEA1 at the nuclear envelope (NE); inset 2 shows the colocalization of ErbB-2 and EEA1 in the nucleus (Nu); inset 3 indicates the colocalization of ErbB-2 and EEA1 in the cytoplasm (Cy). An arrow indicates the source of the inset. (D) MCF-7/HER18 cells were immunostained for EEA1 (green) and ErbB-2 (red) and analyzed by confocal microscopy. Colocalization of EEA1 and ErbB-2 in the nucleus (arrowhead) is indicated in the inset panel. (E) Cytoplasmic and nuclear lysates from MCF-7/HER18 cells were immunoprecipitated with rabbit anti-EEA1 antibody or control rIgG antibody. The immunocomplexes were then analyzed by Western blotting with indicated antibodies. (F) ErbB-2 and importin β1 form complexes with endosomal protein EEA1. Cell extracts from MCF-7/HER18 cells were first immunoprecipitated (1st IP) with antibodies against ErbB-2 or control mouse IgG (mIgG), followed by a second immunoprecipitation (2nd IP) with antibodies against EEA1 or control rIgG. The immunocomplexes were subjected to Western blotting with anti-importin β1 (top), anti-ErbB-2 (middle), and anti-EEA1 (bottom) antibodies. Total cell lysate and single immunoprecipitation with anti-ErbB-2 or mouse IgG antibodies were used as positive controls. The minus sign indicates the absence of antibody. The two lanes marked with asterisks were exposed for a longer period.

    Journal: Molecular and Cellular Biology

    Article Title: Endosomal Transport of ErbB-2: Mechanism for Nuclear Entry of the Cell Surface Receptor †

    doi: 10.1128/MCB.25.24.11005-11018.2005

    Figure Lengend Snippet: Nuclear translocation of ErbB-2 by endosomal sorting. (A) Model for importin β1-mediated ErbB-2 nuclear transport by endosomal sorting. The scale of the diagram does not reflect the relative sizes of different molecules and the subcellular structures. (B) ErbB-2 and importin β1 colocalized in the endosome wall by electron microscopy. An ultrathin section of MCF-7/HER18 cells was immunostained for ErbB-2 (15-nm gold particles, thick arrow) and importin β1 (5-nm gold particles, thin arrow). The arrowhead indicates colocalization of the two proteins in the endosome (E) wall. (C) ErbB-2 and EEA1 colocalization in the cytoplasm and nucleus was demonstrated by electron microscopy. Ultrathin sections from MCF-7/HER18 cells were immunostained for ErbB-2 (5-nm gold particles) and EEA1 (15-nm gold particles). Inset 1 reveals the colocalization of ErbB-2 and EEA1 at the nuclear envelope (NE); inset 2 shows the colocalization of ErbB-2 and EEA1 in the nucleus (Nu); inset 3 indicates the colocalization of ErbB-2 and EEA1 in the cytoplasm (Cy). An arrow indicates the source of the inset. (D) MCF-7/HER18 cells were immunostained for EEA1 (green) and ErbB-2 (red) and analyzed by confocal microscopy. Colocalization of EEA1 and ErbB-2 in the nucleus (arrowhead) is indicated in the inset panel. (E) Cytoplasmic and nuclear lysates from MCF-7/HER18 cells were immunoprecipitated with rabbit anti-EEA1 antibody or control rIgG antibody. The immunocomplexes were then analyzed by Western blotting with indicated antibodies. (F) ErbB-2 and importin β1 form complexes with endosomal protein EEA1. Cell extracts from MCF-7/HER18 cells were first immunoprecipitated (1st IP) with antibodies against ErbB-2 or control mouse IgG (mIgG), followed by a second immunoprecipitation (2nd IP) with antibodies against EEA1 or control rIgG. The immunocomplexes were subjected to Western blotting with anti-importin β1 (top), anti-ErbB-2 (middle), and anti-EEA1 (bottom) antibodies. Total cell lysate and single immunoprecipitation with anti-ErbB-2 or mouse IgG antibodies were used as positive controls. The minus sign indicates the absence of antibody. The two lanes marked with asterisks were exposed for a longer period.

    Article Snippet: The antibodies used in this study were as follows: anti-ErbB-2 (Oncogene); anti-dynamin 2, anti-EPS15, mouse immunoglobulin G1 (IgG1), mouse IgG2a, anticlathrin, and anti-poly(ADP-ribose) polymerase (anti-PARP; BD Biosciences); antiadaptin (Affinity Bioreagents); anti-importin β1 and anticalreticulin (Santa Cruz Biotechnology); anti-EEA1 and antiphosphotyrosine (Upstate Biotechnology Inc.); anti-α-tubulin and anti-Flag (Sigma); anti-green fluorescent protein (anti-GFP; NeoMarkers); and anti-histone H3 (Cell Signaling).

    Techniques: Translocation Assay, Electron Microscopy, Confocal Microscopy, Immunoprecipitation, Western Blot

    Duration of serum FMDV-specific IgG responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Duration of serum FMDV-specific IgG responses. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection

    Numbers of FMDV-specific IgG-secreting cells in bone marrow. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO, or RO containing GS-R (4 μg) on days 1 and 21. Bone marrow cells were prepared 8 weeks after boosting,

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Numbers of FMDV-specific IgG-secreting cells in bone marrow. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO, or RO containing GS-R (4 μg) on days 1 and 21. Bone marrow cells were prepared 8 weeks after boosting,

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay

    Serum FMDV-specific IgG titers. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Sera were collected 2 weeks after boosting, and serum FMDV-specific

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG titers. Mice ( n = 8/group) were s.c. immunized with FMDV antigen plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Sera were collected 2 weeks after boosting, and serum FMDV-specific

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay

    Serum FMDV-specific IgG levels. Mice ( n = 8/group) were s.c. injected with FMDV antigen plus physiological saline, RO, or RO containing GSLS (2, 4, or 6 μg) or GS-R (2, 4, or 6 μg) on days 1 and 21. Sera were collected 3 and 7 days after

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG levels. Mice ( n = 8/group) were s.c. injected with FMDV antigen plus physiological saline, RO, or RO containing GSLS (2, 4, or 6 μg) or GS-R (2, 4, or 6 μg) on days 1 and 21. Sera were collected 3 and 7 days after

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection

    Serum FMDV-specific IgG isotype levels. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological saline

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Rapeseed Oil and Ginseng Saponins Work Synergistically To Enhance Th1 and Th2 Immune Responses Induced by the Foot-and-Mouth Disease Vaccine

    doi: 10.1128/CVI.00127-14

    Figure Lengend Snippet: Serum FMDV-specific IgG isotype levels. Mice ( n = 8/group) were s.c. immunized with FMDV antigen (Ag) plus physiological saline, RO containing GS-R (2, 4, or 6 μg), or ISA 206 on days 1 and 21. Control mice were injected with physiological saline

    Article Snippet: To determine IgG subclasses, 50 μl of HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (1:1,000; Santa Cruz Biotechnology Inc.) was added to each well, and the wells were then incubated for 1 h at 37°C.

    Techniques: Mouse Assay, Injection

    Intracellular interaction between βCTF and TMEM30A. ], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.

    Journal: PLoS ONE

    Article Title: TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

    doi: 10.1371/journal.pone.0200988

    Figure Lengend Snippet: Intracellular interaction between βCTF and TMEM30A. ], α′CTF appeared to be derived from α-secretase cleavage. C, D: COS-7 cells were transfected with APP and the wild-type or KKLN mutant of CFP-TMEM30A. (C) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody to precipitate CFP-TMEM30A. (D) Immunofluorescence analysis. Cells were labeled with APPC15 antibody (green). Scale bar: 20 μm. E: Coimmunoprecipitation analysis of COS-7 cells that were transfected with artificial βCTF, SC100, and wild-type or KKLN mutant of CFP-TMEM30A using control mouse IgG or GFP antibody.

    Article Snippet: For immunoprecipitation, mouse IgG2a (M9144, Sigma-Aldrich) or IgG1 (M3198, Sigma-Aldrich) was used as a control.

    Techniques: Derivative Assay, Transfection, Mutagenesis, Immunofluorescence, Labeling

    TMEM30A interacted with APP at the APP N-terminal domain and Aβ N-terminal sequence. A: Schematic depiction of deletion mutants of APP-Venus used in this study. TM; Transmembrane domain. B: COS-7 cells were transfected with mCherry-TMEM30A and the wild-type or deletion mutant of APP-Venus. After 24 h, the medium was replaced with a fresh medium containing a γ-secretase inhibitor (10 μM DAPT) and further incubated for 24 h. (B) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody (3E6) to precipitate APP-Venus. The arrowhead, arrow, and asterisks represent mature, immature mCherry-TMEM30A, and deletion mutants of APP-Venus, respectively.

    Journal: PLoS ONE

    Article Title: TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

    doi: 10.1371/journal.pone.0200988

    Figure Lengend Snippet: TMEM30A interacted with APP at the APP N-terminal domain and Aβ N-terminal sequence. A: Schematic depiction of deletion mutants of APP-Venus used in this study. TM; Transmembrane domain. B: COS-7 cells were transfected with mCherry-TMEM30A and the wild-type or deletion mutant of APP-Venus. After 24 h, the medium was replaced with a fresh medium containing a γ-secretase inhibitor (10 μM DAPT) and further incubated for 24 h. (B) Coimmunoprecipitation analysis of COS-7 cell lysates using control mouse IgG or GFP antibody (3E6) to precipitate APP-Venus. The arrowhead, arrow, and asterisks represent mature, immature mCherry-TMEM30A, and deletion mutants of APP-Venus, respectively.

    Article Snippet: For immunoprecipitation, mouse IgG2a (M9144, Sigma-Aldrich) or IgG1 (M3198, Sigma-Aldrich) was used as a control.

    Techniques: Sequencing, Transfection, Mutagenesis, Incubation

    TMEM30A interacts with βCTF in endosomes. A: APP and CFP-TMEM30A were cotransfected into COS-7 cells. The effects of TMEM30A expressions on FL-APP and APP-CTFs were analyzed using immunoblotting of cell lysates. 6E10, a monoclonal antibody against N-terminal portion of Aß, was used for ßCTF detection. B: APP, CFP-TMEM30A, and mCherry-ATP8A1 were transfected into COS-7 cells. Coimmunoprecipitation analysis of COS-7 cell lysates was performed using control mouse IgG or GFP antibody (3E6) to precipitate CFP-TMEM30A. C: COS-7 cells were transfected with APP and CFP-TMEM30A. Upper panel: Cells were labeled with Aβ/βCTF-specific antibody (82E1, green) and APP C15 (red). Lower panel: Cells were labeled with the APP C15 (green) and 22C11, (red). Scale bar: 20 μm. D: Analysis of intracellular Aβ accumulation of APP and TMEM30A-transfected COS-7 cells. 48 h after transfection, cells were lysed and the lysate containing 2 mg protein was immunoprecipitated with 82E1. Control was performed by adding indicated amounts of synthetic Aβ40 to the lysis buffer followed by immunoprecipitation. E: Analysis of intracellular sAPPβ accumulation in COS-7 cells transfected with APP and TMEM30A or BACE1. 48 h after transfection, cells were lysed and the lysate containing 2 mg protein was immunoprecipitated with 22C11. Precipitates were analyzed using the sAPPβ-specific antibody.

    Journal: PLoS ONE

    Article Title: TMEM30A is a candidate interacting partner for the β-carboxyl-terminal fragment of amyloid-β precursor protein in endosomes

    doi: 10.1371/journal.pone.0200988

    Figure Lengend Snippet: TMEM30A interacts with βCTF in endosomes. A: APP and CFP-TMEM30A were cotransfected into COS-7 cells. The effects of TMEM30A expressions on FL-APP and APP-CTFs were analyzed using immunoblotting of cell lysates. 6E10, a monoclonal antibody against N-terminal portion of Aß, was used for ßCTF detection. B: APP, CFP-TMEM30A, and mCherry-ATP8A1 were transfected into COS-7 cells. Coimmunoprecipitation analysis of COS-7 cell lysates was performed using control mouse IgG or GFP antibody (3E6) to precipitate CFP-TMEM30A. C: COS-7 cells were transfected with APP and CFP-TMEM30A. Upper panel: Cells were labeled with Aβ/βCTF-specific antibody (82E1, green) and APP C15 (red). Lower panel: Cells were labeled with the APP C15 (green) and 22C11, (red). Scale bar: 20 μm. D: Analysis of intracellular Aβ accumulation of APP and TMEM30A-transfected COS-7 cells. 48 h after transfection, cells were lysed and the lysate containing 2 mg protein was immunoprecipitated with 82E1. Control was performed by adding indicated amounts of synthetic Aβ40 to the lysis buffer followed by immunoprecipitation. E: Analysis of intracellular sAPPβ accumulation in COS-7 cells transfected with APP and TMEM30A or BACE1. 48 h after transfection, cells were lysed and the lysate containing 2 mg protein was immunoprecipitated with 22C11. Precipitates were analyzed using the sAPPβ-specific antibody.

    Article Snippet: For immunoprecipitation, mouse IgG2a (M9144, Sigma-Aldrich) or IgG1 (M3198, Sigma-Aldrich) was used as a control.

    Techniques: Transfection, Labeling, Immunoprecipitation, Lysis

    Involvement of HAMA in FcγR-mediated trogocytosis. ( A ) Effects of HAMA blockade on FcγR-mediated trogocytosis (n = 10). Heparinized whole blood samples were added with or without 1 µl of HAMA inhibitor, TRU block, and then made to react with the PE-labeled anti-CD8α (HIT8a) and FITC-labeled anti-CD15 (H198) Abs. After depletion of erythrocytes, these cells were subjected to FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. Student t -test for paired samples was applied for statistical analysis. ( B ) Difference in the inhibition rates of FcγR-mediated trogocytosis by the HAMA inhibitor between HAMA high sera and HAMA low sera. The serum samples were divided into 2 groups, including HAMA high serum (more than 10 ng/ml, n = 5) and HAMA low serum (less than 10 ng/ml, n = 5). The decrease rates of CD8 + granulocytes by treatment with TRU block were calculated from the data presented in Figure 6A . Student t -test for unpaired samples was applied for statistical analysis.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Involvement of HAMA in FcγR-mediated trogocytosis. ( A ) Effects of HAMA blockade on FcγR-mediated trogocytosis (n = 10). Heparinized whole blood samples were added with or without 1 µl of HAMA inhibitor, TRU block, and then made to react with the PE-labeled anti-CD8α (HIT8a) and FITC-labeled anti-CD15 (H198) Abs. After depletion of erythrocytes, these cells were subjected to FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. Student t -test for paired samples was applied for statistical analysis. ( B ) Difference in the inhibition rates of FcγR-mediated trogocytosis by the HAMA inhibitor between HAMA high sera and HAMA low sera. The serum samples were divided into 2 groups, including HAMA high serum (more than 10 ng/ml, n = 5) and HAMA low serum (less than 10 ng/ml, n = 5). The decrease rates of CD8 + granulocytes by treatment with TRU block were calculated from the data presented in Figure 6A . Student t -test for unpaired samples was applied for statistical analysis.

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Blocking Assay, Labeling, Inhibition

    Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8 + granulocytes. ( A ) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8 + granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. ( B ) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8 + granulocytes. PMNs and PBMCs were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8 + granulocytes. ( A ) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8 + granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. ( B ) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8 + granulocytes. PMNs and PBMCs were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Incubation, Labeling, Inhibition

    Requirement of serum and cell-to-cell interaction with T cells in detection of CD8 + granulocytes. ( A ) mRNA expressions of CD3ε, CD11b, CD8α, and CD8β in granulocytes (CD15 + PMNs) and T cells (CD3 + cells) determined by RT-PCR. These cells were separated from blood samples as described in Materials and Methods . The quality of RNA samples was verified by the expression of GAPDH. ( B ) Requirement of serum for detection of CD8 + granulocytes. PMNs and PBMCs separated from heparinized peripheral blood were mixed in PBS or autologous serum. These cells were incubated with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. ( C ) Requirement of cell-to-cell contact with T cells for detection of CD8 + granulocytes. PMNs and T cells were separated from heparinized blood samples. PMNs were cultured with or without T cells in the autologous serum. In the co-culture wells, PMNs were cultured separately from T cells using the transwell chambers or mixed together with T cells. Subsequently, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. These experiments were repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Requirement of serum and cell-to-cell interaction with T cells in detection of CD8 + granulocytes. ( A ) mRNA expressions of CD3ε, CD11b, CD8α, and CD8β in granulocytes (CD15 + PMNs) and T cells (CD3 + cells) determined by RT-PCR. These cells were separated from blood samples as described in Materials and Methods . The quality of RNA samples was verified by the expression of GAPDH. ( B ) Requirement of serum for detection of CD8 + granulocytes. PMNs and PBMCs separated from heparinized peripheral blood were mixed in PBS or autologous serum. These cells were incubated with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. ( C ) Requirement of cell-to-cell contact with T cells for detection of CD8 + granulocytes. PMNs and T cells were separated from heparinized blood samples. PMNs were cultured with or without T cells in the autologous serum. In the co-culture wells, PMNs were cultured separately from T cells using the transwell chambers or mixed together with T cells. Subsequently, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. These experiments were repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Labeling, Cell Culture, Co-Culture Assay

    Characterization of serum factors that contribute to FcγR-mediated trogocytosis. ( A ) Heat instability of serum factors that contribute to FcγR-mediated trogocytosis (n = 5). PMNs and PBMCs were mixed in sera, which had been heated at 56°C for 30 min, or sera without heat inactivation. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. Student t -test for paired samples was applied for statistical analysis. ( B ) Molecular weight range of serum factors that contribute to FcγR-mediated trogocytosis. PMNs and PBMCs were mixed in sera, which had been fractionated into those with molecular weight of more than 100 kDa or less than 100 kDa. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. This experiment was repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Characterization of serum factors that contribute to FcγR-mediated trogocytosis. ( A ) Heat instability of serum factors that contribute to FcγR-mediated trogocytosis (n = 5). PMNs and PBMCs were mixed in sera, which had been heated at 56°C for 30 min, or sera without heat inactivation. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. Student t -test for paired samples was applied for statistical analysis. ( B ) Molecular weight range of serum factors that contribute to FcγR-mediated trogocytosis. PMNs and PBMCs were mixed in sera, which had been fractionated into those with molecular weight of more than 100 kDa or less than 100 kDa. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. This experiment was repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Labeling, Molecular Weight

    Bystander transfer of CD3 and TCRαβ molecules accompanied by CD8 translocation from T cells to granulocytes. Heparinized blood samples were pre-incubated with the unlabeled anti-CD8α Ab (HIT8a) or mouse IgG1, followed by depletion of erythrocytes, and then re-suspended in PBS. The cells were made to react with the PE-labeled anti-CD3 (UCHT-1) and FITC-labeled anti-CD15 (H198) Abs, or the FITC-labeled anti-TCRαβ (WT31) and PE-labeled anti-CD15 (H198) Abs, followed by serving for FCM. PE or FITC-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Bystander transfer of CD3 and TCRαβ molecules accompanied by CD8 translocation from T cells to granulocytes. Heparinized blood samples were pre-incubated with the unlabeled anti-CD8α Ab (HIT8a) or mouse IgG1, followed by depletion of erythrocytes, and then re-suspended in PBS. The cells were made to react with the PE-labeled anti-CD3 (UCHT-1) and FITC-labeled anti-CD15 (H198) Abs, or the FITC-labeled anti-TCRαβ (WT31) and PE-labeled anti-CD15 (H198) Abs, followed by serving for FCM. PE or FITC-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Translocation Assay, Incubation, Labeling

    Detection of CD8 + granulocytes and correlation to CD8 + monocytes. ( A ) Detection of CD8 + granulocytes in human peripheral blood samples. Heparinized whole blood samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS, and then allowed to react with the FITC-labeled anti-CD15 Ab (H198). PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. The cells gated in R1 were regarded as PMNs. Among the PMNs, granulocytes were characterized by the high level of expression of CD15 (gated in R2). The cells in both R1 and R2 gates were examined for the expression of CD8α. ( B ) Correlation of CD8 + granulocytes and CD8 + monocytes (n = 32, r = 0.92, p = 9.46×10 −12 in Pearson correlation test).

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Detection of CD8 + granulocytes and correlation to CD8 + monocytes. ( A ) Detection of CD8 + granulocytes in human peripheral blood samples. Heparinized whole blood samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS, and then allowed to react with the FITC-labeled anti-CD15 Ab (H198). PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. The cells gated in R1 were regarded as PMNs. Among the PMNs, granulocytes were characterized by the high level of expression of CD15 (gated in R2). The cells in both R1 and R2 gates were examined for the expression of CD8α. ( B ) Correlation of CD8 + granulocytes and CD8 + monocytes (n = 32, r = 0.92, p = 9.46×10 −12 in Pearson correlation test).

    Article Snippet: Isotype-matched mouse IgG1 (MOPC-21, BD Pharmingen), IgG2a (DAK-GO5, Dako), and IgM (MM-30, BioLegend, San Diego, CA) served as controls.

    Techniques: Labeling, Expressing

    Control of renal inflammation by gut microbiota in female lpr mice. a Serum level of IgG2a ( n = 7 mice per group; *** P

    Journal: Microbiome

    Article Title: Control of lupus nephritis by changes of gut microbiota

    doi: 10.1186/s40168-017-0300-8

    Figure Lengend Snippet: Control of renal inflammation by gut microbiota in female lpr mice. a Serum level of IgG2a ( n = 7 mice per group; *** P

    Article Snippet: Serum IgG, IgA, IgG2a, and IL-10 concentrations were determined with mouse IgG, IgA, IgG2a (Bethyl Laboratories), and IL-10 (Biolegend) ELISA kits, respectively, according to the manufacturers’ instructions.

    Techniques: Mouse Assay

    Lactobacillus spp. protect female lpr mice from LN. a Time-dependent changes of fecal microbiota upon PBS or Lactobacillus ( Lacto ) treatment ( n = 4 mice per group). b Level of anti-dsDNA IgG in the blood of 10-week-old mice ( n = 7 mice per group; ** P

    Journal: Microbiome

    Article Title: Control of lupus nephritis by changes of gut microbiota

    doi: 10.1186/s40168-017-0300-8

    Figure Lengend Snippet: Lactobacillus spp. protect female lpr mice from LN. a Time-dependent changes of fecal microbiota upon PBS or Lactobacillus ( Lacto ) treatment ( n = 4 mice per group). b Level of anti-dsDNA IgG in the blood of 10-week-old mice ( n = 7 mice per group; ** P

    Article Snippet: Serum IgG, IgA, IgG2a, and IL-10 concentrations were determined with mouse IgG, IgA, IgG2a (Bethyl Laboratories), and IL-10 (Biolegend) ELISA kits, respectively, according to the manufacturers’ instructions.

    Techniques: Mouse Assay

    TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes. (A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control IgG, at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2 -ΔΔCT , where ΔΔC T is the average difference between the siTACSTD2 ΔC T and siControl ΔC T .

    Journal: PLoS Pathogens

    Article Title: Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma

    doi: 10.1371/journal.ppat.1006916

    Figure Lengend Snippet: TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes. (A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control IgG, at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2 -ΔΔCT , where ΔΔC T is the average difference between the siTACSTD2 ΔC T and siControl ΔC T .

    Article Snippet: The antibodies used include mouse anti-TACSTD2 (Santa Cruz), goat anti-TACSTD2, biotinylated goat anti-TACSTD2 and goat anti-E-cadherin, mouse IgG1 isotype control, mouse IgG2A isotype control, and normal goat IgG control (R & D systems), mouse anti-CLDN1 (Abnova), rabbit anti-CLDN1 and mouse anti-OCLN (Life Technologies), rabbit anti-OCLN (Abcam), rabbit anti-CD81 and mouse anti-ZO-1 (Thermo Scientific), mouse anti-SR-B1 (BD transduction laboratories), mouse anti-CD81 (BD Pharmingen), mouse anti-JAM-A (Hycult Biotech), mouse anti-HCV core (Anogen), rabbit anti-PKC substrate (Cell Signaling Technologies), and rabbit anti-alpha tubulin (Millipore).

    Techniques: Transfection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    TACSTD2 expression and interaction with HCV-entry cofactors in hepatoma cells. (A) Western blot analysis of TACSTD2 expression in parental Huh7.5 cells and TACSTD2-overexpressing Huh7.5 cells (TACSTD2). TACSTD2 expression was below the detection limit of Western blot in parental cells (lane 1) but was visible in TACSTD2-overexpressing cells as a broad band due to its poly-glycoslyated state (lane 2). Enzymatic deglycosylation resulted in the protein migration as a single band of 37KD, as seen in lane 4. (B) Immunofluorescence detection of TACSTD2 (green) in parental and TACSTD2-overexpressing Huh7.5 cells. No TACSTD2 could be visualized in parental Huh7.5 (top panel), whereas in stably transfected cells, TACSTD2 was localized both in the cytoplasm and along the cellular membrane (lower panel). (C) Detection of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells by immunoprecipitation with an anti-TACSTD2 antibody without deglycosylation. (D) Detection of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells by immunoprecipitation with an anti-TACSTD2 antibody followed by deglycosylation. (E) Phosphorylation of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates were immunoprecipitated with anti-TACSTD2 antibody and then deglycosylated. Phosphorylated TACSTD2 was detected using an anti-PKC substrate-specific antibody; total TACSTD2 was detected using biotinylated anti-TACSTD2 antibody. Phosphorylated TACSTD2 was detected in both parental and TACSTD2-overexpressing Huh7.5 cells. (F) Co-immunoprecipitation of CLDN1 and OCLN by anti-TACSTD2 antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Lysates prepared from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-TACSTD2 antibody or control IgG, deglycosylated, and probed with antibodies specific for TACSTD2, CLDN1, and OCLN. (G) Reciprocal co-immunoprecipitation of TACSTD2 by anti-CLDN1 antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Cell lysates were immunoprecipitated with anti-CLDN1 antibody or control IgG. After deglycosylation, total TACSTD2 was detected using biotinylated anti-TACSTD2 antibody, phosphorylated TACSTD2 was detected using an anti-PKC substrate-specific antibody, and CLDN1 was detected using an anti-CLDN1 specific antibody. (H) Reciprocal co-immunoprecipitation of TACSTD2 by anti-OCLN antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Cell lysates were immunoprecipitated with anti-OCLN antibody or control IgG, deglycosylated, and probed with biotinylated anti-TACSTD2 antibody, anti-PKC substrate-specific antibody to detect phosphorylated TACSTD2, and anti-OCLN specific antibody.

    Journal: PLoS Pathogens

    Article Title: Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma

    doi: 10.1371/journal.ppat.1006916

    Figure Lengend Snippet: TACSTD2 expression and interaction with HCV-entry cofactors in hepatoma cells. (A) Western blot analysis of TACSTD2 expression in parental Huh7.5 cells and TACSTD2-overexpressing Huh7.5 cells (TACSTD2). TACSTD2 expression was below the detection limit of Western blot in parental cells (lane 1) but was visible in TACSTD2-overexpressing cells as a broad band due to its poly-glycoslyated state (lane 2). Enzymatic deglycosylation resulted in the protein migration as a single band of 37KD, as seen in lane 4. (B) Immunofluorescence detection of TACSTD2 (green) in parental and TACSTD2-overexpressing Huh7.5 cells. No TACSTD2 could be visualized in parental Huh7.5 (top panel), whereas in stably transfected cells, TACSTD2 was localized both in the cytoplasm and along the cellular membrane (lower panel). (C) Detection of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells by immunoprecipitation with an anti-TACSTD2 antibody without deglycosylation. (D) Detection of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells by immunoprecipitation with an anti-TACSTD2 antibody followed by deglycosylation. (E) Phosphorylation of TACSTD2 in parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates were immunoprecipitated with anti-TACSTD2 antibody and then deglycosylated. Phosphorylated TACSTD2 was detected using an anti-PKC substrate-specific antibody; total TACSTD2 was detected using biotinylated anti-TACSTD2 antibody. Phosphorylated TACSTD2 was detected in both parental and TACSTD2-overexpressing Huh7.5 cells. (F) Co-immunoprecipitation of CLDN1 and OCLN by anti-TACSTD2 antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Lysates prepared from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-TACSTD2 antibody or control IgG, deglycosylated, and probed with antibodies specific for TACSTD2, CLDN1, and OCLN. (G) Reciprocal co-immunoprecipitation of TACSTD2 by anti-CLDN1 antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Cell lysates were immunoprecipitated with anti-CLDN1 antibody or control IgG. After deglycosylation, total TACSTD2 was detected using biotinylated anti-TACSTD2 antibody, phosphorylated TACSTD2 was detected using an anti-PKC substrate-specific antibody, and CLDN1 was detected using an anti-CLDN1 specific antibody. (H) Reciprocal co-immunoprecipitation of TACSTD2 by anti-OCLN antibody in parental and TACSTD2-overexpressing Huh7.5 cells (lanes 1, 2). Cell lysates were immunoprecipitated with anti-OCLN antibody or control IgG, deglycosylated, and probed with biotinylated anti-TACSTD2 antibody, anti-PKC substrate-specific antibody to detect phosphorylated TACSTD2, and anti-OCLN specific antibody.

    Article Snippet: The antibodies used include mouse anti-TACSTD2 (Santa Cruz), goat anti-TACSTD2, biotinylated goat anti-TACSTD2 and goat anti-E-cadherin, mouse IgG1 isotype control, mouse IgG2A isotype control, and normal goat IgG control (R & D systems), mouse anti-CLDN1 (Abnova), rabbit anti-CLDN1 and mouse anti-OCLN (Life Technologies), rabbit anti-OCLN (Abcam), rabbit anti-CD81 and mouse anti-ZO-1 (Thermo Scientific), mouse anti-SR-B1 (BD transduction laboratories), mouse anti-CD81 (BD Pharmingen), mouse anti-JAM-A (Hycult Biotech), mouse anti-HCV core (Anogen), rabbit anti-PKC substrate (Cell Signaling Technologies), and rabbit anti-alpha tubulin (Millipore).

    Techniques: Expressing, Western Blot, Migration, Immunofluorescence, Stable Transfection, Transfection, Immunoprecipitation