mouse igg1 Search Results


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  • 99
    Vector Laboratories biotinylated secondary antibodies
    Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or <t>biotinylated</t> secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p
    Biotinylated Secondary Antibodies, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 10949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    LI-COR irdye 800cw goat anti mouse igg h l
    Western blotting with infrared dye–labeled <t>anti-IgG</t> nanobodies. (a) A twofold dilution series of Xenopus egg extract was analyzed by SDS-PAGE and Western blotting. The indicated rabbit polyclonal antibodies were used to detect Nups. These primary antibodies were then decorated via either <t>IRDye</t> 800–labeled goat anti–rabbit polyclonal IgG (1:5,000; LI-COR Biosciences) or anti–rabbit IgG nanobody TP897 (10 nM). Blots were analyzed with an Odyssey Infrared Imaging System (LI-COR Biosciences). (b) Left: A twofold dilution series of HeLa cell lysate was analyzed by SDS-PAGE and Western blotting. The indicated mouse IgG1 mAbs were decorated via either IRDye 800–labeled goat anti–mouse polyclonal IgG (1:1,340, 5 nM; LI-COR Biosciences) or anti–mouse IgG1 Fc nanobody TP1107 (5 nM). Right: A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225. It was then detected via IRDye 800–labeled goat anti-mouse polyclonal IgG (5 nM), anti–mouse IgG1 Fc nanobody TP1107 (5 nM), anti–mouse IgG1 Fab nanobody TP886 (5 nM), anti–mouse κ chain nanobody TP1170 (2.5 nM), or a combination of TP1107 and TP886 or TP1107 and TP1170. Blue pixels indicate signal saturation. (c) A dilution series of filamentous bacteriophages was blotted and probed with an anti–minor coat protein pIII mouse IgG2a mAb. It was then decorated via either IRDye 800–labeled goat anti-mouse polyclonal IgG (2.5 nM) or anti–mouse κ chain nanobody TP1170 (2.5 nM). (d) Dual-color Western blotting. A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225 and rabbit anti-Nup54 polyclonal antibody. These primary antibodies were then detected via IRDye 800–labeled goat anti–rabbit polyclonal IgG and IRDye 680–labeled goat anti–mouse polyclonal IgG. Alternatively, they were detected with TP1107 coupled to IRDye 680 and TP897 coupled to IRDye 800.
    Irdye 800cw Goat Anti Mouse Igg H L, supplied by LI-COR, used in various techniques. Bioz Stars score: 99/100, based on 2660 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse <t>IgG</t> was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher donkey anti mouse igg h l highly cross adsorbed secondary antibody
    In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse <t>IgG</t> was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p
    Donkey Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 6700 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore mouse igg3
    TE-7 stains cells below the epidermis in normal skin tissue. Human dermis was collected from breast tissue, fixed, and paraffin-embedded. Slides were cut and stained with an <t>IgG1</t> isotype control or the TE-7 antibody as described in Materials and Methods.
    Mouse Igg3, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mouse immunoglobulin g
    DHX32 promotes transcription of VEGFA in CRC cells. (a) SW480 stable cells were transfected with β-catenin/TCF luciferase reporter gene pTOPFLASH or mutated pFOPFLASH, pRL-TK as internal control. The mean ± SD of a representative result of three independent experiments is shown. (b) Real-time RT-PCR analyses of VEGFA expression in SW480 cells with DHX32 depletion or overexpression. (c) Endogenous VEGFA protein levels in SW480 cells with DHX32 overexpression or depletion were detected by immunoblotting. (d) SW480 stable cells with depletion DHX32 were transfected with β-catenin and DHX32-overexpressed stable cells were transfected with control siRNA or siRNA against β-catenin. VEGFA protein levels in condition medium were quantified by ELISA analysis. (e) Representative PCR gel of ChIP assays showing binding of β-catenin to the VEGF promoter over the <t>IgG</t> control. Immunoprecipitate was carried out using an antibody to β-catenin. Nonimmunoprecipitated chromatin was used as an “input” control, and an IgG antibody control was performed on all occasions. The PCR primers were amplified in the − 262 to − 101 region of the VEGF promoter. ** P
    Mouse Immunoglobulin G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher anti mouse igg
    In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and <t>β-actin</t> proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS <t>IgG)</t> or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P
    Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher goat anti mouse igg2a
    β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and <t>IgG2a-specific</t> antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.
    Goat Anti Mouse Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg whole molecule peroxidase antibody
    Binding of <t>IgG</t> and <t>C3b</t> with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher alexa fluor 488 goat anti mouse igg
    FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by <t>Alexa</t> <t>fluor</t> 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P
    Alexa Fluor 488 Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology goat anti mouse igg hrp
    Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by <t>HRP</t> conjugated anti-mouse <t>IgG.</t>
    Goat Anti Mouse Igg Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 4245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology mouse immunoglobulin g
    BRG1 and GR do not associate with the promoter in the absence of NF1 binding. Cells from stable cell lines containing Wt (pLS-Wt) or mNF1 constructs were treated without or with DEX for 1 h and analyzed by ChIP assay with BRG1 and GR antibodies. The cells were sonicated and immunoprecipitated with nonspecific (rabbit <t>IgG)</t> or BRG1 antibodies (A) or nonspecific (mouse IgG) or GR antibodies (C). The MMTV promoter sequence was detected by PCR using oligo-38 and 32 P-end-labeled oligo-22. The PCR products were run on nondenaturing polyacrylamide gels and exposed to PhosphorImager screens for further analysis. The association of BRG1 (B) or GR (D) with Wt and NF1 templates was quantified from two independent experiments and by setting the association with the wild-type promoter in the presence of DEX to 100.
    Mouse Immunoglobulin G, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg2b
    PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total <t>IgG</t> and IgG subgroups <t>(IgG1,</t> <t>IgG2a,</t> and <t>IgG2b)</t> in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.
    Anti Mouse Igg2b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti mouse igg
    <t>IRGM1</t> activates PI3K-Rac1 signaling pathway. B16 cells were transfected with either control or IRGM1siRNA ( a ) or transduced with either GFP or IRGM1-GFP lentiviral vectors ( b – h ). ( a , b ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. ( c ) Co-localization of PIK3CA with IRGM1 was determined by immunofluorescence staining in IRGM1 overexpressing B16 cells (Upper panel: GFP; lower panel: IRGM1-GFP; green: IRGM1-GFP/GFP; red: PI3KCA; blue: nuclei). ( d ) Co-IP study was performed to further confirm the co-localization of IRGM1 and PIK3CA. Anti-IRGM1 polyclonal antibody or non-immune <t>IgG</t> (control) were used to pull down IRGM1 from total cell lysates. Anti-PIK3CA monoclonal antibody was used to detect participated PIK3CA. GFP and IRGM1 overexpressing B16 cells were transfected with either control or PIK3CA siRNA ( e – h ). ( e ) PIK3CA levels were measured by western blot. ( f ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. The migration ( g ) and invasion ( h ) ability of GFP and IRGM1 overexpressing B16 cells were evaluated by using boyden chamber. Data represent three independent experiments. (Unpaired t-test; **p
    Anti Mouse Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4035 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher igg1
    Antibody response and chemokine immunomediators in serum. A. Total concentration of IgM, IgG, <t>IgG1,</t> IgG2a and IgG3 antibodies, and B. CxCL1, CxCL2, CCL5, TNFα and IFNγ, which were quantified by ELISA two weeks post-infection. Statistics: unpaired t test with Welch’s correction between infected groups, Total IgM, p = 0.0457; Total IgG p = 0.0133; Total IgG1 p
    Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jackson Immuno peroxidase affinipure goat anti mouse igg
    Reactivity of pooled Ugandan serum samples and a vaccinated mouse serum pool against synthetic peptide series I. ( A ) High, ( B ) medium-high, ( C ) medium-low, ( D ) low titer sera pool. Each pool consists of equal aliquots of 9–10 individual sera. The geometric mean anti-SE36 <t>IgG</t> titers of the individual samples are in Table S2 . The patterns of reactivity for 18 individual sera are shown in Fig. S2 . Serum samples were diluted 800-fold. Secondary antibody was peroxidase-conjugated goat IgG fraction to human IgG (whole molecule) (55220; Cappel ICN Pharmaceuticals Inc, Aurora, OH) diluted 1∶2000. ( E ) Pooled serum from five mice used at 1∶1,600. Secondary antibody was peroxidase conjugated <t>affiniPure</t> goat anti-mouse IgG antibody (H+L) (115-035-166; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1∶5000. All sera were tested for ELISA at least four times. Error bars reflect standard deviation. Reactivity of malaria naïve Japanese serum and naïve mouse serum are shown in Fig. S2 .
    Peroxidase Affinipure Goat Anti Mouse Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 3302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology normal mouse igg
    MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against <t>CREB,</t> RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and <t>IgG</t> (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
    Normal Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 3651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg
    The amino-terminal glycine is required for efficient VLP production and Pr Gag membrane targeting. (A) VLP production. pGag-HA and mutants were stably transfected into COS-1 cells. Cell and virion lysates were analyzed by Western blot using an anti-HA antibody. The Western blots were subjected to quantitative fluorochemical analysis as described in the text. This experiment was repeated three times, with representative results shown. (B) Cellular distribution of Pr Gag . Cells stably transfected with VLP constructs were grown on coverslips, fixed, and incubated with an anti-HA Ig followed by incubation with <t>Alexa</t> Fluor 488-conjugated anti-mouse Ig. Images were collected using a confocal microscope.
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson mouse igg2a
    Anti-OPN antibodies suppress established chronic CHS. Mice were sensitized on day 0 with 7% TNCB in acetone or acetone as control. CHS was elicited with 1% TNCB or acetone as indicated in A . TNCB sensitized animals were injected i.p. with 400 μg of anti-OPN monoclonal antibody or isotype matched control antibody on days 10, 13, and 16 ( A and B ). Ear swelling was measured daily beginning on day 5. TNCB <t>IgG:</t> n = 5, TNCB anti-OPN: n = 5, acetone: n = 8 mice. (Statistically significant, unpaired t -test: ∗ P
    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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    93
    Santa Cruz Biotechnology goat anti mouse igg
    Sec22b concentrates on VTVs and co-localizes with p58 and Sar1 on their surface ( A ) Samples containing hepatic whole-cell lysate (WCL), VTVs, ER and Golgi (each containing30 μ g of protein) were separated by SDS/PAGE (12 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against GOS28, <t>calnexin,</t> and Rab11. For details, see the Experimental section. Protein detection was by ECL. ( B ) Samples of ER and VTVs (30 μ g of protein each) were separated by SDS/PAGE (5–15 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against Sec22b, Ykt6, ApoB100 and albumin. Protein detection was by ECL. ( C ) Immunoelectron microscopy of VTVs utilizing the negative-staining technique. VTVs were adsorbed on formvar-carbon-coated nickel grids and were treated with: (i) anti-rabbit pre-immune <t>IgG;</t> (ii) rabbit polyclonal anti-Sec22b antibodies detected with anti-(rabbit IgG) labelled with 15 nm gold particles; (iii) mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles and anti-Sar1 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles (arrows show co-localization of Sec22b with Sar1); and (iv) anti-p58 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles and mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles (arrows show co-localization of Sec22b with p58). Scale bars, 100 nm.
    Goat Anti Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories biotinylated anti mouse igg
    Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal <t>IgG</t> (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using <t>biotinylated</t> anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P
    Biotinylated Anti Mouse Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 3254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare anti mouse igg
    NKD1 and NKD2 are targets of tankyrase. a Schematic diagram of NKD1/2 showing the N-terminal myristoylation site. The shaded box indicates the conserved EFX (EF-hand containing) domain that binds DVL. Alignment of the tankyrase-binding site in human NKD1 and NKD2. Identical amino acids are in black. b Immunoblot analysis showing NKD2 is stabilized in RNF146 siRNA-treated HEK293T cells. c , d Endogenous tankyrase and <t>GFP-NKD2</t> coimmunoprecipitate, dependent on the NKD2 tankyrase-binding site. Immunoblot analysis of HEK293T cells transfected with GFP, GFP-NKD2 WT, or GFP-NKD2 Mut, immunoprecipitated with c tankyrase or d GFP antibodies, and probed with the indicated antibodies. e Immunoblot analysis showing that NKD1 is stabilized upon treatment of HepG2 cells with tankyrase inhibitor Ti8. f Immunoblot analysis showing that HA-NKD1 coimmunoprecitates with tankyrase. Immunoblot of HEK293T cells transfected with HA-NKD1, immunoprecipitated with control or TNKS <t>IgG,</t> and probed with the indicated antibodies
    Anti Mouse Igg, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 4476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SouthernBiotech goat anti mouse igg2a
    E. fuscus Ig preferentially use lambda light chains . (Upper) Varying volumes of E. fuscus serum (expressed in μl) and swine <t>IgG</t> (expressed in μg) or (Lower) 0.03 μl of E. fuscus , BALB/c, or horse serum, as indicated, were fractionated using protein L-magnetic microbeads. The unbound ( λ ) and protein-L-binding ( κ ) material was reduced and then resolved using SDS-PAGE, followed by visualization using silver stain. Boxed areas represent individual IgH and IgL chains.
    Goat Anti Mouse Igg2a, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 97/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or biotinylated secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p

    Journal: Molecular Brain

    Article Title: Region-specific astrogliosis: differential vessel formation contributes to different patterns of astrogliosis in the cortex and striatum

    doi: 10.1186/s13041-020-00642-0

    Figure Lengend Snippet: Avastin attenuates astrogliosis and blood vessel formation in the injured cortex. a, c Cortical sections were obtained from mice in which Avastin or PBS was infused into the ventricle 14 d post-ATP injection. GFAP and CoL1A1 were visualized with Alexa-488 and Alexa-594 conjugated secondary antibodies ( a ) or biotinylated secondary antibodies and DAB-based color reaction ( c ). b Relative fluorescence intensity of GFAP and CoL1A1 were measured using ZEN software and plotted. Values are means ± SEMs for animals treated with saline ( n = 4) or Avastin ( n = 3) (*, p

    Article Snippet: The sections were rinsed three times with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

    Techniques: Mouse Assay, Injection, Fluorescence, Software

    Western blotting with infrared dye–labeled anti-IgG nanobodies. (a) A twofold dilution series of Xenopus egg extract was analyzed by SDS-PAGE and Western blotting. The indicated rabbit polyclonal antibodies were used to detect Nups. These primary antibodies were then decorated via either IRDye 800–labeled goat anti–rabbit polyclonal IgG (1:5,000; LI-COR Biosciences) or anti–rabbit IgG nanobody TP897 (10 nM). Blots were analyzed with an Odyssey Infrared Imaging System (LI-COR Biosciences). (b) Left: A twofold dilution series of HeLa cell lysate was analyzed by SDS-PAGE and Western blotting. The indicated mouse IgG1 mAbs were decorated via either IRDye 800–labeled goat anti–mouse polyclonal IgG (1:1,340, 5 nM; LI-COR Biosciences) or anti–mouse IgG1 Fc nanobody TP1107 (5 nM). Right: A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225. It was then detected via IRDye 800–labeled goat anti-mouse polyclonal IgG (5 nM), anti–mouse IgG1 Fc nanobody TP1107 (5 nM), anti–mouse IgG1 Fab nanobody TP886 (5 nM), anti–mouse κ chain nanobody TP1170 (2.5 nM), or a combination of TP1107 and TP886 or TP1107 and TP1170. Blue pixels indicate signal saturation. (c) A dilution series of filamentous bacteriophages was blotted and probed with an anti–minor coat protein pIII mouse IgG2a mAb. It was then decorated via either IRDye 800–labeled goat anti-mouse polyclonal IgG (2.5 nM) or anti–mouse κ chain nanobody TP1170 (2.5 nM). (d) Dual-color Western blotting. A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225 and rabbit anti-Nup54 polyclonal antibody. These primary antibodies were then detected via IRDye 800–labeled goat anti–rabbit polyclonal IgG and IRDye 680–labeled goat anti–mouse polyclonal IgG. Alternatively, they were detected with TP1107 coupled to IRDye 680 and TP897 coupled to IRDye 800.

    Journal: The Journal of Cell Biology

    Article Title: A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

    doi: 10.1083/jcb.201709115

    Figure Lengend Snippet: Western blotting with infrared dye–labeled anti-IgG nanobodies. (a) A twofold dilution series of Xenopus egg extract was analyzed by SDS-PAGE and Western blotting. The indicated rabbit polyclonal antibodies were used to detect Nups. These primary antibodies were then decorated via either IRDye 800–labeled goat anti–rabbit polyclonal IgG (1:5,000; LI-COR Biosciences) or anti–rabbit IgG nanobody TP897 (10 nM). Blots were analyzed with an Odyssey Infrared Imaging System (LI-COR Biosciences). (b) Left: A twofold dilution series of HeLa cell lysate was analyzed by SDS-PAGE and Western blotting. The indicated mouse IgG1 mAbs were decorated via either IRDye 800–labeled goat anti–mouse polyclonal IgG (1:1,340, 5 nM; LI-COR Biosciences) or anti–mouse IgG1 Fc nanobody TP1107 (5 nM). Right: A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225. It was then detected via IRDye 800–labeled goat anti-mouse polyclonal IgG (5 nM), anti–mouse IgG1 Fc nanobody TP1107 (5 nM), anti–mouse IgG1 Fab nanobody TP886 (5 nM), anti–mouse κ chain nanobody TP1170 (2.5 nM), or a combination of TP1107 and TP886 or TP1107 and TP1170. Blue pixels indicate signal saturation. (c) A dilution series of filamentous bacteriophages was blotted and probed with an anti–minor coat protein pIII mouse IgG2a mAb. It was then decorated via either IRDye 800–labeled goat anti-mouse polyclonal IgG (2.5 nM) or anti–mouse κ chain nanobody TP1170 (2.5 nM). (d) Dual-color Western blotting. A twofold dilution series of Xenopus egg extract was blotted and probed with anti-Nup62 mouse IgG1 mAb A225 and rabbit anti-Nup54 polyclonal antibody. These primary antibodies were then detected via IRDye 800–labeled goat anti–rabbit polyclonal IgG and IRDye 680–labeled goat anti–mouse polyclonal IgG. Alternatively, they were detected with TP1107 coupled to IRDye 680 and TP897 coupled to IRDye 800.

    Article Snippet: Polyclonal goat anti–mouse IgG coupled to IRDye 800CW (925-32210; LI-COR Biosciences) was used to detect primary mouse antibodies at a dilution of 1:1,340 (5 nM).

    Techniques: Western Blot, Labeling, SDS Page, Imaging

    In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse IgG was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p

    Journal: PLoS ONE

    Article Title: Prostaglandin E2 (PGE2) Exerts Biphasic Effects on Human Tendon Stem Cells

    doi: 10.1371/journal.pone.0087706

    Figure Lengend Snippet: In vivo expression of non-tenocyte markers PPARγ, collagen type II, and osteocalcin in rats implanted with hTSCs treated with various concentrations of PGE 2 and their respective hematoxylin and eosin (H E) stained tissue sections. hTSCs cultured with three concentrations (0.1, 10, and 100 ng/ml) of PGE 2 were implanted subcutaneously into rats; later, immunohistochemical and histological analyses were performed on tissue sections. For the immunohistochemical staining, fixed tissue sections were incubated with mouse anti-human PPARγ antibody, mouse anti-collagen type II (Collagen II) antibody, or mouse anti-human osteocalcin antibody. Cy3-conjugated goat anti-mouse IgG was then used to detect primary binding. Nuclei were stained with Hoechst (blue). Expression levels of PPARγ, collagen type II, and osteocalcin (red) were lower in cells treated with 0.1 ng/ml PGE 2 ( A–C ) than those treated with the higher concentrations (10 and 100 ng/ml) of PGE 2 ( E–G, I–K ). H E staining was also performed on tissue sections ( D, H, L ). More cells (black dots; see insets in D , H , and L ) were observed in tissues implanted with hTSCs that had been treated with high concentrations of PGE 2 in culture ( H, L ). Specifically, at 100 ng/ml ( L ), cells were concentrated in a specific region (triangle). Additionally, semi-quantification of the stained cells was performed by counting immuno-positive cells and calculating percentage staining ( M ) (*p

    Article Snippet: The cells were then washed three times with PBS, followed by incubation with Cy3-conjugated goat anti-mouse IgG (1∶500; Invitrogen, Cat. # A10521) secondary antibody at room temperature for 2 hrs.

    Techniques: In Vivo, Expressing, Staining, Cell Culture, Immunohistochemistry, Incubation, Binding Assay

    TE-7 stains cells below the epidermis in normal skin tissue. Human dermis was collected from breast tissue, fixed, and paraffin-embedded. Slides were cut and stained with an IgG1 isotype control or the TE-7 antibody as described in Materials and Methods.

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: An Immunohistochemical Method for Identifying Fibroblasts in Formalin-fixed, Paraffin-embedded Tissue

    doi: 10.1369/jhc.7A7287.2007

    Figure Lengend Snippet: TE-7 stains cells below the epidermis in normal skin tissue. Human dermis was collected from breast tissue, fixed, and paraffin-embedded. Slides were cut and stained with an IgG1 isotype control or the TE-7 antibody as described in Materials and Methods.

    Article Snippet: Isotype control antibodies were mouse IgG (I-2000; Vector Laboratories), mouse IgG1 (CBL600; Chemicon, Temecula, CA), mouse IgG2a (CBL601; Chemicon), mouse IgG3 (M9019; Sigma-Aldrich, St Louis, MO), mouse IgM (PP50; Chemicon), and rabbit IgG (011-000-003; Jackson Immunoresearch Laboratories).

    Techniques: Staining

    DHX32 promotes transcription of VEGFA in CRC cells. (a) SW480 stable cells were transfected with β-catenin/TCF luciferase reporter gene pTOPFLASH or mutated pFOPFLASH, pRL-TK as internal control. The mean ± SD of a representative result of three independent experiments is shown. (b) Real-time RT-PCR analyses of VEGFA expression in SW480 cells with DHX32 depletion or overexpression. (c) Endogenous VEGFA protein levels in SW480 cells with DHX32 overexpression or depletion were detected by immunoblotting. (d) SW480 stable cells with depletion DHX32 were transfected with β-catenin and DHX32-overexpressed stable cells were transfected with control siRNA or siRNA against β-catenin. VEGFA protein levels in condition medium were quantified by ELISA analysis. (e) Representative PCR gel of ChIP assays showing binding of β-catenin to the VEGF promoter over the IgG control. Immunoprecipitate was carried out using an antibody to β-catenin. Nonimmunoprecipitated chromatin was used as an “input” control, and an IgG antibody control was performed on all occasions. The PCR primers were amplified in the − 262 to − 101 region of the VEGF promoter. ** P

    Journal: EBioMedicine

    Article Title: DHX32 Promotes Angiogenesis in Colorectal Cancer Through Augmenting β-catenin Signaling to Induce Expression of VEGFA

    doi: 10.1016/j.ebiom.2017.03.012

    Figure Lengend Snippet: DHX32 promotes transcription of VEGFA in CRC cells. (a) SW480 stable cells were transfected with β-catenin/TCF luciferase reporter gene pTOPFLASH or mutated pFOPFLASH, pRL-TK as internal control. The mean ± SD of a representative result of three independent experiments is shown. (b) Real-time RT-PCR analyses of VEGFA expression in SW480 cells with DHX32 depletion or overexpression. (c) Endogenous VEGFA protein levels in SW480 cells with DHX32 overexpression or depletion were detected by immunoblotting. (d) SW480 stable cells with depletion DHX32 were transfected with β-catenin and DHX32-overexpressed stable cells were transfected with control siRNA or siRNA against β-catenin. VEGFA protein levels in condition medium were quantified by ELISA analysis. (e) Representative PCR gel of ChIP assays showing binding of β-catenin to the VEGF promoter over the IgG control. Immunoprecipitate was carried out using an antibody to β-catenin. Nonimmunoprecipitated chromatin was used as an “input” control, and an IgG antibody control was performed on all occasions. The PCR primers were amplified in the − 262 to − 101 region of the VEGF promoter. ** P

    Article Snippet: DHX32 proteins were detected using rabbit anti-DHX32 antibody followed by Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Life Technologies). β-catenin was detected using mouse anti-β-catenin antibody followed by Alexa Fluor 488-conjugated donkey antibody to mouse immunoglobulin G (Life Technologies).

    Techniques: Transfection, Luciferase, Quantitative RT-PCR, Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Amplification

    In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and β-actin proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS IgG) or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P

    Journal: PLoS ONE

    Article Title: Regulation of Cyclooxygenase-2 Expression by Heat: A Novel Aspect of Heat Shock Factor 1 Function in Human Cells

    doi: 10.1371/journal.pone.0031304

    Figure Lengend Snippet: In vitro and in vivo binding of HSF1 to the COX-2 promoter. ( A ) Nucleotide sequence of the 100 bp (from −2579 to −2480) DNA fragment of the COX-2 promoter region including binding sites for the GATA and HSF1 transcription factors. The 100 bp fragment was amplified by PCR, 32 P-labeled and used as probe for gel-shift analysis shown in B and C . ( B ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), whole-cell extracts were analyzed for HSF DNA-binding activity by EMSA in a 3% polyacrylamide gel using the probe described in ( A ). Position of HSF-DNA binding complex (HSF), constitutive HSE binding activity (CHBA) and non specific protein-DNA interactions (NS) are shown (upper panel). In parallel samples whole-cell extracts were analyzed for levels of HSF1 and β-actin proteins by Western blot (lower panels). Arrow indicates the position of the low-mobility phosphorylated HSF1 isoform. ( C ) Specificity of HSF1-DNA binding complexes. Whole-cell extracts from HUVECs subjected to heat shock at 43°C for 40 min were preincubated with different dilutions of anti-HSF1 polyclonal antibodies for 15 min before supershift assay. Position of HSF, CHBA and NS are indicated as in B . ( D,E ) HUVECs were subjected to heat shock at 43°C or left untreated (control). After 20 and 40 min at 43°C (HS) or at the indicated times during recovery at 37°C (recovery), recruitment of HSF1 to the COX-2 and HSP70 promoters was analyzed by ChIP assay. ( D ) ChIP-enriched DNAs using preimmune serum (IP NS IgG) or anti-HSF1 serum (IP anti-HSF1), as well as input DNAs (INPUT) were prepared, and DNA fragments of the COX-2 gene (−2629 to −2420) and HSP70 gene (−262 to −70) were amplified by PCR. ( E ) Quantification of ChIP assay shown in ( D ). Samples from at least three independent experiments were analyzed by real time PCR. Relative promoter occupancy is expressed as fold induction of control arbitrarily set to a value of 1. Error bars indicate ± S.D. * = P

    Article Snippet: After blocking with 5% skim milk solution, membranes were incubated with rabbit polyclonal anti-HSF1, (Santa Cruz Biotechnology), antibodies, or monoclonal anti-COX-2 (SC-19999, Santa Cruz Biotechnology), anti-HSP70 (Stressgene), and anti-β-actin (Sigma) antibodies followed by decoration with peroxidase-labeled anti-rabbit or anti-mouse IgG respectively (Super Signal detection kit, Pierce).

    Techniques: In Vitro, In Vivo, Binding Assay, Sequencing, Amplification, Polymerase Chain Reaction, Labeling, Electrophoretic Mobility Shift Assay, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

    Journal: Journal of Virology

    Article Title: Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product

    doi: 10.1128/JVI.76.6.2899-2911.2002

    Figure Lengend Snippet: β-Gal-specific antibody titers in mice immunized with AdβGal or with AdβGal-transduced muscle cells. Mice received one i.m. injection with either 10 9 PFU of AdβGal (Ad) or 5 × 10 4 AdβGal-expressing DC (DC/Ad), myoblasts (Myo/Ad), or EC (EC/Ad). Mice injected with untransduced DC, myoblasts, or EC (DC, Myo, or EC) were used as negative controls. Mice were bled once a week, and β-Gal-specific antibody titers were measured by ELISA. (A) Generation of β-Gal-specific IgG antibodies as a function of time. (B) Levels of IgG1- and IgG2a-specific antibodies at day 42. (C) Kinetics of IgG1 and IgG2a β-Gal-specific antibodies after immunization with AdβGal-transduced DC.

    Article Snippet: Different anti-mouse Ig's were used: goat-anti-mouse IgG (Amersham Pharmacia), goat anti-mouse IgG1 (Caltag), and goat anti-mouse IgG2a (Caltag).

    Techniques: Mouse Assay, Injection, Expressing, Enzyme-linked Immunosorbent Assay

    Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Journal: Frontiers in Immunology

    Article Title: Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation

    doi: 10.3389/fimmu.2020.00462

    Figure Lengend Snippet: Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Article Snippet: C3b was detected in bacterial lysates ran on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) without heating under non-reducing conditions (without adding 2-mercaptoethanol), transferred to nitrocellulose membrane (MDI), and probed with mouse anti-human C3b monoclonal antibodies (Thermo Fisher, Cat. No. MA1-70054) followed with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (Sigma, Cat. No. A4416).

    Techniques: Binding Assay, Incubation, Purification

    FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by Alexa fluor 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P

    Journal: Nucleic Acids Research

    Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

    doi: 10.1093/nar/gky403

    Figure Lengend Snippet: FANCJ-R707C is a separation-of-function mutant that partially rescues sensitivity to the replication stress inducing agents aphidicolin or telomestatin but fails to restore cisplatin resistance. ( A ) Schematic diagram of green fluorescence protein (GFP)-FANCJ recombinant protein expressed in fancj −/− cells for genetic complementation assays. ( B ) Western blot analysis of whole cell lysate protein (40 μg) from fancj −/− DT40 cells transfected with plasmid encoding (pEGFP-Vector), pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C), or pEGFP-FANCJ-H396D (FANCJ-H396D). Protein was detected with antibody against FANCJ or actin (as a loading control, 10% loaded). (C-E) Cell survival assays, as measured by methyl cellulose colony formation, for the indicated fancj −/− cells expressing mutant or wild-type FANCJ proteins. Cells were exposed to the indicated concentrations of CisPt ( C ), APH ( D ) or TMS ( E ). Filled square, fancj −/− cells transfected with FANCJ-WT; open square, fancj −/− cells transfected with pEGFP-Vector; filled triangle, fancj −/− cells transfected with FANCJ-R707C; open cross- fancj −/− cells transfected with FANCJ-H396D. (F, G) FANCJ-R707C restores normal replication restart after APH exposure. Schematic representation of the protocol used to track DNA replication fibers is shown in Panel F. Cells were pulse-chased with CldU ( red label ), and then labeled with IdU ( green label ) for the indicated times. ( G ) Representative images of fluorescently-labeled DNA fibers from the indicated cell lines treated with DMSO or APH. ( H ) Box and whiskers graphs indicating the 10–90 percentile of the IdU tract length (μm) for ongoing forks. The data, presented as mean ± standard error of the mean (s.e.m.), are based on the measurement of at least 100 DNA fibers from two independent experiments. ( I – N ), DNA damage, as marked by immunofluorescent detection of γ-H2AX foci, in the indicated fancj −/− transfected cell lines. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to: CisPt (1 μM) (I, J); APH (200 nM) (K, L); TMS (5 μM) (M, N). Immunofluorescence detection of γ-H2AX foci by Alexa fluor 488 is shown along with DAPI, or merged (DAPI and Alexa fluor 488). Quantitative analyses of γ-H2AX foci are shown with S.D. (ns-not significant; ** P

    Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

    Techniques: Mutagenesis, Fluorescence, Recombinant, Western Blot, Transfection, Plasmid Preparation, Expressing, Labeling, Immunofluorescence

    FANCJ-R707C fails to suppress single-stranded DNA or Rad51 persistence in cells exposed to CisPt. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to CisPt (1 μM) ( A, B ) or APH (200 nM) ( C, D ). Note that for BrdU staining, cells were pre-labeled with BrdU prior to drug exposure as described in Materials and Methods. (A, C) Immunofluorescent BrdU foci (A) or Rad51 foci (C) were detected by Alexa fluor 488 and are shown along with DAPI, or merged (DAPI and Alexa fluor 488). (B, D) Quantitative analyses of BrdU foci (B) or Rad51 foci (D) are shown with S.D. indicated by error bars.

    Journal: Nucleic Acids Research

    Article Title: A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair

    doi: 10.1093/nar/gky403

    Figure Lengend Snippet: FANCJ-R707C fails to suppress single-stranded DNA or Rad51 persistence in cells exposed to CisPt. fancj −/− cells transfected with pEGFP-Vector, pEGFP-FANCJ-WT (FANCJ-WT), pEGFP-FANCJ-R707C (FANCJ-R707C) or pEGFP-FANCJ-H396D (FANCJ-H396D) were exposed for 14 h to CisPt (1 μM) ( A, B ) or APH (200 nM) ( C, D ). Note that for BrdU staining, cells were pre-labeled with BrdU prior to drug exposure as described in Materials and Methods. (A, C) Immunofluorescent BrdU foci (A) or Rad51 foci (C) were detected by Alexa fluor 488 and are shown along with DAPI, or merged (DAPI and Alexa fluor 488). (B, D) Quantitative analyses of BrdU foci (B) or Rad51 foci (D) are shown with S.D. indicated by error bars.

    Article Snippet: After four washes in PBS with 0.1% Tween 20, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or Alexa Fluor 633 goat anti-mouse IgG (1:500, Invitrogen) for 1 h at room temperature.

    Techniques: Transfection, Plasmid Preparation, BrdU Staining, Labeling

    Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by HRP conjugated anti-mouse IgG.

    Journal: Neurobiology of aging

    Article Title: MultiTEP Platform-based DNA Vaccines for alpha-Synucleinopathies: Pre-clinical Evaluation of Immunogenicity and Therapeutic Potency

    doi: 10.1016/j.neurobiolaging.2017.08.006

    Figure Lengend Snippet: Schematic representation of DNA hα-Syn vaccine constructs and expression in transfected CHO cells. (A ) Strategy for cloning genes encoding several hα-Syn B cell epitope/s fused with MultiTEP into the pVAX1 vector and schematic representation of PV-1947D, PV-1948D, PV-1949D, and PV-1950D constructs. ( B–D ) The expression of PV-1947D ( B ), PV-1948D ( C ), PV-1949D ( D ), and PV-1950D ( B–D ) and secretion of protein was demonstrated by WB of conditioned media of transiently transfected CHO cells. Proteins were visualized by staining with anti-hα-Syn 85–99 Abs ( A ), anti-α-hSyn 109–126 Abs ( B ), and anti-α-hSyn 126–140 Abs ( C ) followed by HRP conjugated anti-mouse IgG.

    Article Snippet: Membranes were stained with purified anti-hα-Syn85–99 , anti-hα-Syn109–126 and anti-hα-Syn126–140 antibodies at concentration 1µg/ml and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology).

    Techniques: Construct, Expressing, Transfection, Clone Assay, Plasmid Preparation, Western Blot, Staining

    BRG1 and GR do not associate with the promoter in the absence of NF1 binding. Cells from stable cell lines containing Wt (pLS-Wt) or mNF1 constructs were treated without or with DEX for 1 h and analyzed by ChIP assay with BRG1 and GR antibodies. The cells were sonicated and immunoprecipitated with nonspecific (rabbit IgG) or BRG1 antibodies (A) or nonspecific (mouse IgG) or GR antibodies (C). The MMTV promoter sequence was detected by PCR using oligo-38 and 32 P-end-labeled oligo-22. The PCR products were run on nondenaturing polyacrylamide gels and exposed to PhosphorImager screens for further analysis. The association of BRG1 (B) or GR (D) with Wt and NF1 templates was quantified from two independent experiments and by setting the association with the wild-type promoter in the presence of DEX to 100.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Factor 1 Is Required for Both Hormone-Dependent Chromatin Remodeling and Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter

    doi: 10.1128/MCB.23.3.887-898.2003

    Figure Lengend Snippet: BRG1 and GR do not associate with the promoter in the absence of NF1 binding. Cells from stable cell lines containing Wt (pLS-Wt) or mNF1 constructs were treated without or with DEX for 1 h and analyzed by ChIP assay with BRG1 and GR antibodies. The cells were sonicated and immunoprecipitated with nonspecific (rabbit IgG) or BRG1 antibodies (A) or nonspecific (mouse IgG) or GR antibodies (C). The MMTV promoter sequence was detected by PCR using oligo-38 and 32 P-end-labeled oligo-22. The PCR products were run on nondenaturing polyacrylamide gels and exposed to PhosphorImager screens for further analysis. The association of BRG1 (B) or GR (D) with Wt and NF1 templates was quantified from two independent experiments and by setting the association with the wild-type promoter in the presence of DEX to 100.

    Article Snippet: Ten micrograms of antibody (anti-BRG1 [Santa Cruz Biotech], anti-GR [made from myeloma cells; FIGR; American Type Culture Collection]), mouse immunoglobulin G (IgG; Santa Cruz Biotech), or rabbit IgG (Santa Cruz Biotech) was added to tubes containing 900 μl of chromatin solution.

    Techniques: Binding Assay, Stable Transfection, Construct, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Labeling

    Antibody responses in sera of mice immunized with M2e-FP-1 and M2e-FP-2 proteins . Mice were immunized with M2e-FP-1 and M2e-FP-2 proteins, or PBS as a control, and sera were collected at the indicated time points post-immunization to detect M2e-FP-specific IgG (A), IgG1 (B), and IgG2a (C) antibodies by ELISA. The antibody titers were expressed as the endpoint dilutions that remain positively detectable, and presented as mean ± SD of 5 mice in each group.

    Journal: Microbes and Infection

    Article Title: Highly conserved M2e and hemagglutinin epitope-based recombinant proteins induce protection against influenza virus infection

    doi: 10.1016/j.micinf.2017.08.010

    Figure Lengend Snippet: Antibody responses in sera of mice immunized with M2e-FP-1 and M2e-FP-2 proteins . Mice were immunized with M2e-FP-1 and M2e-FP-2 proteins, or PBS as a control, and sera were collected at the indicated time points post-immunization to detect M2e-FP-specific IgG (A), IgG1 (B), and IgG2a (C) antibodies by ELISA. The antibody titers were expressed as the endpoint dilutions that remain positively detectable, and presented as mean ± SD of 5 mice in each group.

    Article Snippet: After 4 washes, the plates were incubated with HRP-conjugated anti-mouse IgG, anti-mouse IgG1, and anti-mouse IgG2a (1:5,000, Santa Cruz), respectively, for 1 h at 37 °C.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total IgG and IgG subgroups (IgG1, IgG2a, and IgG2b) in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.

    Journal: Frontiers in Microbiology

    Article Title: PA0833 Is an OmpA C-Like Protein That Confers Protection Against Pseudomonas aeruginosa Infection

    doi: 10.3389/fmicb.2018.01062

    Figure Lengend Snippet: PA0833 stimulates protective immunity in a P. aeruginosa sepsis model. (A) Comparison of total IgG and IgG subgroups (IgG1, IgG2a, and IgG2b) in the mice ( n = 5) immunized with PA0833. Serum was obtained at 7 days after the final immunization, and the levels of total IgG and IgG subgroups were expressed as the means of log 2 titers. Multiple comparisons among different groups were analyzed using one-way ANOVA. Data are shown as the means ± SD. (B) BALB/c mice ( n = 10) were immunized with PA0833 plus an Al(OH) 3 adjuvant and challenged with PAO1 at 7.0 × 10 7 CFUs/mouse by intravenous injection. The survival rate was monitored for 14 days. The P -values were calculated using the Mantel–Cox log-rank test. (C,D) Efficacy of immunization with PA0833 on the spread of P. aeruginosa . The number of viable bacteria in the blood, liver, and spleen of mice ( n = 10) at 1 and 3 days post-infection are shown. Data are presented in box and whisker plots, and the medians are shown. Differences were compared to determine their significance using Student’s t -test.

    Article Snippet: Diluted serum samples were used as the primary antibodies, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Sigma).

    Techniques: Mouse Assay, Injection, Infection, Whisker Assay

    IRGM1 activates PI3K-Rac1 signaling pathway. B16 cells were transfected with either control or IRGM1siRNA ( a ) or transduced with either GFP or IRGM1-GFP lentiviral vectors ( b – h ). ( a , b ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. ( c ) Co-localization of PIK3CA with IRGM1 was determined by immunofluorescence staining in IRGM1 overexpressing B16 cells (Upper panel: GFP; lower panel: IRGM1-GFP; green: IRGM1-GFP/GFP; red: PI3KCA; blue: nuclei). ( d ) Co-IP study was performed to further confirm the co-localization of IRGM1 and PIK3CA. Anti-IRGM1 polyclonal antibody or non-immune IgG (control) were used to pull down IRGM1 from total cell lysates. Anti-PIK3CA monoclonal antibody was used to detect participated PIK3CA. GFP and IRGM1 overexpressing B16 cells were transfected with either control or PIK3CA siRNA ( e – h ). ( e ) PIK3CA levels were measured by western blot. ( f ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. The migration ( g ) and invasion ( h ) ability of GFP and IRGM1 overexpressing B16 cells were evaluated by using boyden chamber. Data represent three independent experiments. (Unpaired t-test; **p

    Journal: Scientific Reports

    Article Title: IRGM1 enhances B16 melanoma cell metastasis through PI3K-Rac1 mediated epithelial mesenchymal transition

    doi: 10.1038/srep12357

    Figure Lengend Snippet: IRGM1 activates PI3K-Rac1 signaling pathway. B16 cells were transfected with either control or IRGM1siRNA ( a ) or transduced with either GFP or IRGM1-GFP lentiviral vectors ( b – h ). ( a , b ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. ( c ) Co-localization of PIK3CA with IRGM1 was determined by immunofluorescence staining in IRGM1 overexpressing B16 cells (Upper panel: GFP; lower panel: IRGM1-GFP; green: IRGM1-GFP/GFP; red: PI3KCA; blue: nuclei). ( d ) Co-IP study was performed to further confirm the co-localization of IRGM1 and PIK3CA. Anti-IRGM1 polyclonal antibody or non-immune IgG (control) were used to pull down IRGM1 from total cell lysates. Anti-PIK3CA monoclonal antibody was used to detect participated PIK3CA. GFP and IRGM1 overexpressing B16 cells were transfected with either control or PIK3CA siRNA ( e – h ). ( e ) PIK3CA levels were measured by western blot. ( f ) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. The migration ( g ) and invasion ( h ) ability of GFP and IRGM1 overexpressing B16 cells were evaluated by using boyden chamber. Data represent three independent experiments. (Unpaired t-test; **p

    Article Snippet: Antibodies used for Western blot include anti-IRGM1 pAb (AbMart), anti-PIK3CA pAb (proteintech), anti-Rac1 pAb (Cell Signaling), anti-tubulin (Sigma), anti-mouse IgG and anti-rabbit IgG (Cell Signaling).

    Techniques: Transfection, Transduction, Activity Assay, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Migration

    Antibody response and chemokine immunomediators in serum. A. Total concentration of IgM, IgG, IgG1, IgG2a and IgG3 antibodies, and B. CxCL1, CxCL2, CCL5, TNFα and IFNγ, which were quantified by ELISA two weeks post-infection. Statistics: unpaired t test with Welch’s correction between infected groups, Total IgM, p = 0.0457; Total IgG p = 0.0133; Total IgG1 p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Pre-treatment with Lactobacillus plantarum prevents severe pathogenesis in mice infected with Leptospira interrogans and may be associated with recruitment of myeloid cells

    doi: 10.1371/journal.pntd.0005870

    Figure Lengend Snippet: Antibody response and chemokine immunomediators in serum. A. Total concentration of IgM, IgG, IgG1, IgG2a and IgG3 antibodies, and B. CxCL1, CxCL2, CCL5, TNFα and IFNγ, which were quantified by ELISA two weeks post-infection. Statistics: unpaired t test with Welch’s correction between infected groups, Total IgM, p = 0.0457; Total IgG p = 0.0133; Total IgG1 p

    Article Snippet: ELISA Quantification of total mouse immunoglobulin concentration IgM, IgG, IgG1, IgG2a, IgG3 in mouse serum was done using Ready-Set-Go ELISA kits (eBioscience).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection

    Reactivity of pooled Ugandan serum samples and a vaccinated mouse serum pool against synthetic peptide series I. ( A ) High, ( B ) medium-high, ( C ) medium-low, ( D ) low titer sera pool. Each pool consists of equal aliquots of 9–10 individual sera. The geometric mean anti-SE36 IgG titers of the individual samples are in Table S2 . The patterns of reactivity for 18 individual sera are shown in Fig. S2 . Serum samples were diluted 800-fold. Secondary antibody was peroxidase-conjugated goat IgG fraction to human IgG (whole molecule) (55220; Cappel ICN Pharmaceuticals Inc, Aurora, OH) diluted 1∶2000. ( E ) Pooled serum from five mice used at 1∶1,600. Secondary antibody was peroxidase conjugated affiniPure goat anti-mouse IgG antibody (H+L) (115-035-166; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1∶5000. All sera were tested for ELISA at least four times. Error bars reflect standard deviation. Reactivity of malaria naïve Japanese serum and naïve mouse serum are shown in Fig. S2 .

    Journal: PLoS ONE

    Article Title: Protective Epitopes of the Plasmodium falciparum SERA5 Malaria Vaccine Reside in Intrinsically Unstructured N-Terminal Repetitive Sequences

    doi: 10.1371/journal.pone.0098460

    Figure Lengend Snippet: Reactivity of pooled Ugandan serum samples and a vaccinated mouse serum pool against synthetic peptide series I. ( A ) High, ( B ) medium-high, ( C ) medium-low, ( D ) low titer sera pool. Each pool consists of equal aliquots of 9–10 individual sera. The geometric mean anti-SE36 IgG titers of the individual samples are in Table S2 . The patterns of reactivity for 18 individual sera are shown in Fig. S2 . Serum samples were diluted 800-fold. Secondary antibody was peroxidase-conjugated goat IgG fraction to human IgG (whole molecule) (55220; Cappel ICN Pharmaceuticals Inc, Aurora, OH) diluted 1∶2000. ( E ) Pooled serum from five mice used at 1∶1,600. Secondary antibody was peroxidase conjugated affiniPure goat anti-mouse IgG antibody (H+L) (115-035-166; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1∶5000. All sera were tested for ELISA at least four times. Error bars reflect standard deviation. Reactivity of malaria naïve Japanese serum and naïve mouse serum are shown in Fig. S2 .

    Article Snippet: After washing with PBS/T, peroxidase-conjugated goat IgG fraction to human IgG (whole molecule) (55220; Cappel ICN Pharmaceuticals Inc, Aurora, OH) diluted 1∶2000; or horseradish peroxidase-conjugated rabbit anti-human IgG antibody (A8792; Sigma-Aldrich Corp., St. Louis, MO) diluted 1∶2000; or peroxidase conjugated affiniPure goat anti-mouse IgG antibody (H+L) (115-035-166; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1∶5000 in 5% skim milk in PBS/T was added to the plates and incubated at 37°C for 1 hour.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.

    Journal: Nucleic Acids Research

    Article Title: Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements

    doi: 10.1093/nar/gks1365

    Figure Lengend Snippet: MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.

    Article Snippet: Reagents α-RFX5 was from Rockland, α-NFY-A (H-209) (sc-10779), α-NFYA (G-2) (sc-17753), α-NFY-B (FL-207) (sc-13045), α-CREB (C-21) (sc-186), α-RNA Polymerase II (N-20) (sc-899), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology, α-TBP (ab28175) and α-H3 (ab1791) were from Abcam, α-acetyl H3 (06-599), α-acetyl H4 (06-866), α-H3K4me2 (07-030), α-H3K4me3 (07-473), α-H3S10-Phos (06-570), α-PP2Ac (Cat.# 05-421) and α-CREB (17-600) were from Millipore, α-PP2Ac (#2028) was from Cell Signaling, α-β-tubulin (T4026) was from Sigma-Aldrich, α-GFP was from Minotech, α-JellyRed (α-KillerRed, Cat.# AB962) was from Evrogen, mouse monoclonal α-myc antibody was secreted from the hybridoma cell line Myc-9E10.2 ( ) and anti-CIITA antibody was described earlier ( ).

    Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The DRA -LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. ( A ) DRA -LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA + ) and HeLa cell lines shown in Figure 2 D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2 B. ( B ) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. ( C ) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.

    Journal: Nucleic Acids Research

    Article Title: Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements

    doi: 10.1093/nar/gks1365

    Figure Lengend Snippet: The DRA -LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. ( A ) DRA -LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA + ) and HeLa cell lines shown in Figure 2 D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2 B. ( B ) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. ( C ) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.

    Article Snippet: Reagents α-RFX5 was from Rockland, α-NFY-A (H-209) (sc-10779), α-NFYA (G-2) (sc-17753), α-NFY-B (FL-207) (sc-13045), α-CREB (C-21) (sc-186), α-RNA Polymerase II (N-20) (sc-899), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology, α-TBP (ab28175) and α-H3 (ab1791) were from Abcam, α-acetyl H3 (06-599), α-acetyl H4 (06-866), α-H3K4me2 (07-030), α-H3K4me3 (07-473), α-H3S10-Phos (06-570), α-PP2Ac (Cat.# 05-421) and α-CREB (17-600) were from Millipore, α-PP2Ac (#2028) was from Cell Signaling, α-β-tubulin (T4026) was from Sigma-Aldrich, α-GFP was from Minotech, α-JellyRed (α-KillerRed, Cat.# AB962) was from Evrogen, mouse monoclonal α-myc antibody was secreted from the hybridoma cell line Myc-9E10.2 ( ) and anti-CIITA antibody was described earlier ( ).

    Techniques: Derivative Assay, Expressing, Immunoprecipitation, Western Blot

    The amino-terminal glycine is required for efficient VLP production and Pr Gag membrane targeting. (A) VLP production. pGag-HA and mutants were stably transfected into COS-1 cells. Cell and virion lysates were analyzed by Western blot using an anti-HA antibody. The Western blots were subjected to quantitative fluorochemical analysis as described in the text. This experiment was repeated three times, with representative results shown. (B) Cellular distribution of Pr Gag . Cells stably transfected with VLP constructs were grown on coverslips, fixed, and incubated with an anti-HA Ig followed by incubation with Alexa Fluor 488-conjugated anti-mouse Ig. Images were collected using a confocal microscope.

    Journal: Journal of Virology

    Article Title: Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function

    doi: 10.1128/JVI.76.16.8485-8493.2002

    Figure Lengend Snippet: The amino-terminal glycine is required for efficient VLP production and Pr Gag membrane targeting. (A) VLP production. pGag-HA and mutants were stably transfected into COS-1 cells. Cell and virion lysates were analyzed by Western blot using an anti-HA antibody. The Western blots were subjected to quantitative fluorochemical analysis as described in the text. This experiment was repeated three times, with representative results shown. (B) Cellular distribution of Pr Gag . Cells stably transfected with VLP constructs were grown on coverslips, fixed, and incubated with an anti-HA Ig followed by incubation with Alexa Fluor 488-conjugated anti-mouse Ig. Images were collected using a confocal microscope.

    Article Snippet: The cells were then incubated with anti-HA antibody followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Oreg.).

    Techniques: Stable Transfection, Transfection, Western Blot, Construct, Incubation, Microscopy

    Anti-OPN antibodies suppress established chronic CHS. Mice were sensitized on day 0 with 7% TNCB in acetone or acetone as control. CHS was elicited with 1% TNCB or acetone as indicated in A . TNCB sensitized animals were injected i.p. with 400 μg of anti-OPN monoclonal antibody or isotype matched control antibody on days 10, 13, and 16 ( A and B ). Ear swelling was measured daily beginning on day 5. TNCB IgG: n = 5, TNCB anti-OPN: n = 5, acetone: n = 8 mice. (Statistically significant, unpaired t -test: ∗ P

    Journal: The American Journal of Pathology

    Article Title: Antigen-Specific Induction of Osteopontin Contributes to the Chronification of Allergic Contact Dermatitis

    doi: 10.2353/ajpath.2010.090488

    Figure Lengend Snippet: Anti-OPN antibodies suppress established chronic CHS. Mice were sensitized on day 0 with 7% TNCB in acetone or acetone as control. CHS was elicited with 1% TNCB or acetone as indicated in A . TNCB sensitized animals were injected i.p. with 400 μg of anti-OPN monoclonal antibody or isotype matched control antibody on days 10, 13, and 16 ( A and B ). Ear swelling was measured daily beginning on day 5. TNCB IgG: n = 5, TNCB anti-OPN: n = 5, acetone: n = 8 mice. (Statistically significant, unpaired t -test: ∗ P

    Article Snippet: The anti-human integrin antibodies: αv β3 (LM609), αv β5 (P1FG), β1 (P4G11), α4 (9F10), α9 β1 (Y9A2), and CD45RO (UCHL1) were obtained from Chemicon International (Schwalbach, Germany); CD44s (SFF-2) and CD44v6 (VFF-18) from Bender MedSystems (Vienna, Austria); CD4 (RPA-T4), α4 (9F10), IFN-γ-R (GIR-94), and isotype control antibodies (mouse IgM (G155-228)), mouse IgG1 (107.3), and mouse IgG2a (MOPC-21)) were from BD Pharmingen (Heidelberg, Germany).

    Techniques: Mouse Assay, Injection

    Sec22b concentrates on VTVs and co-localizes with p58 and Sar1 on their surface ( A ) Samples containing hepatic whole-cell lysate (WCL), VTVs, ER and Golgi (each containing30 μ g of protein) were separated by SDS/PAGE (12 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against GOS28, calnexin, and Rab11. For details, see the Experimental section. Protein detection was by ECL. ( B ) Samples of ER and VTVs (30 μ g of protein each) were separated by SDS/PAGE (5–15 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against Sec22b, Ykt6, ApoB100 and albumin. Protein detection was by ECL. ( C ) Immunoelectron microscopy of VTVs utilizing the negative-staining technique. VTVs were adsorbed on formvar-carbon-coated nickel grids and were treated with: (i) anti-rabbit pre-immune IgG; (ii) rabbit polyclonal anti-Sec22b antibodies detected with anti-(rabbit IgG) labelled with 15 nm gold particles; (iii) mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles and anti-Sar1 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles (arrows show co-localization of Sec22b with Sar1); and (iv) anti-p58 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles and mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles (arrows show co-localization of Sec22b with p58). Scale bars, 100 nm.

    Journal: The Biochemical journal

    Article Title: The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi

    doi: 10.1042/BJ20100336

    Figure Lengend Snippet: Sec22b concentrates on VTVs and co-localizes with p58 and Sar1 on their surface ( A ) Samples containing hepatic whole-cell lysate (WCL), VTVs, ER and Golgi (each containing30 μ g of protein) were separated by SDS/PAGE (12 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against GOS28, calnexin, and Rab11. For details, see the Experimental section. Protein detection was by ECL. ( B ) Samples of ER and VTVs (30 μ g of protein each) were separated by SDS/PAGE (5–15 % gel), transblotted on to a nitrocellulose membrane and probed with specific antibodies against Sec22b, Ykt6, ApoB100 and albumin. Protein detection was by ECL. ( C ) Immunoelectron microscopy of VTVs utilizing the negative-staining technique. VTVs were adsorbed on formvar-carbon-coated nickel grids and were treated with: (i) anti-rabbit pre-immune IgG; (ii) rabbit polyclonal anti-Sec22b antibodies detected with anti-(rabbit IgG) labelled with 15 nm gold particles; (iii) mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles and anti-Sar1 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles (arrows show co-localization of Sec22b with Sar1); and (iv) anti-p58 antibodies detected with anti-(rabbit IgG) labelled with 10 nm gold particles and mouse anti-Sec22b antibodies detected with anti-(mouse IgG) labelled with 15 nm gold particles (arrows show co-localization of Sec22b with p58). Scale bars, 100 nm.

    Article Snippet: Goat anti-calnexin, anti-Rab11, goat anti-(rabbit IgG), goat anti-(mouse IgG) and goat anti-(rabbit IgG) antibodies conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology.

    Techniques: SDS Page, Immuno-Electron Microscopy, Negative Staining

    Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal IgG (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using biotinylated anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P

    Journal: Journal of Clinical Investigation

    Article Title: Myocardial protection from ischemia/reperfusion injury by endogenous and exogenous HGF

    doi:

    Figure Lengend Snippet: Adverse effects of neutralization of endogenous HGF on the ischemia/reperfusion injury model. ( a ) Specificity of the neutralizing antibody to HGF. Plasma from a rat with ischemia/reperfusion injury was immunoprecipitated with normal IgG (lane 1) or anti–rat HGF IgG (lane 2), and immunoreactive proteins were detected by Western blot, using biotinylated anti–rat HGF IgG. ( b ) Immunohistochemical staining of infarcted hearts with α-sarcomeric actin to depict the infarct area and its quantification. Anti–rat HGF IgG ( n = 10) or normal IgG ( n = 10) was injected 20 minutes before coronary occlusion, and every 12 hours after reperfusion. Forty-eight hours after operation, rats were killed and histological and biochemical analyses were made. Arrowheads indicate the α-sarcomeric actin–negative infarct area (original magnification, ×40). A P

    Article Snippet: After blocking, the membrane was sequentially incubated with anti–phospho-p44/p42 mitogen-activated protein kinase (ERK-1/2) antibody (E10; New England BioLabs Inc., Beverly, Massachusetts, USA), biotinylated anti-mouse IgG (Vector Laboratories), horseradish peroxidase–conjugated streptavidin (Amersham Pharmacia Biotech UK, Little Chalfont, United Kingdom), and an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech UK).

    Techniques: Neutralization, Immunoprecipitation, Western Blot, Immunohistochemistry, Staining, Injection

    NKD1 and NKD2 are targets of tankyrase. a Schematic diagram of NKD1/2 showing the N-terminal myristoylation site. The shaded box indicates the conserved EFX (EF-hand containing) domain that binds DVL. Alignment of the tankyrase-binding site in human NKD1 and NKD2. Identical amino acids are in black. b Immunoblot analysis showing NKD2 is stabilized in RNF146 siRNA-treated HEK293T cells. c , d Endogenous tankyrase and GFP-NKD2 coimmunoprecipitate, dependent on the NKD2 tankyrase-binding site. Immunoblot analysis of HEK293T cells transfected with GFP, GFP-NKD2 WT, or GFP-NKD2 Mut, immunoprecipitated with c tankyrase or d GFP antibodies, and probed with the indicated antibodies. e Immunoblot analysis showing that NKD1 is stabilized upon treatment of HepG2 cells with tankyrase inhibitor Ti8. f Immunoblot analysis showing that HA-NKD1 coimmunoprecitates with tankyrase. Immunoblot of HEK293T cells transfected with HA-NKD1, immunoprecipitated with control or TNKS IgG, and probed with the indicated antibodies

    Journal: Nature Communications

    Article Title: Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation

    doi: 10.1038/s41467-017-02363-w

    Figure Lengend Snippet: NKD1 and NKD2 are targets of tankyrase. a Schematic diagram of NKD1/2 showing the N-terminal myristoylation site. The shaded box indicates the conserved EFX (EF-hand containing) domain that binds DVL. Alignment of the tankyrase-binding site in human NKD1 and NKD2. Identical amino acids are in black. b Immunoblot analysis showing NKD2 is stabilized in RNF146 siRNA-treated HEK293T cells. c , d Endogenous tankyrase and GFP-NKD2 coimmunoprecipitate, dependent on the NKD2 tankyrase-binding site. Immunoblot analysis of HEK293T cells transfected with GFP, GFP-NKD2 WT, or GFP-NKD2 Mut, immunoprecipitated with c tankyrase or d GFP antibodies, and probed with the indicated antibodies. e Immunoblot analysis showing that NKD1 is stabilized upon treatment of HepG2 cells with tankyrase inhibitor Ti8. f Immunoblot analysis showing that HA-NKD1 coimmunoprecitates with tankyrase. Immunoblot of HEK293T cells transfected with HA-NKD1, immunoprecipitated with control or TNKS IgG, and probed with the indicated antibodies

    Article Snippet: Immunoblot analysis Immunoblots were incubated separately with the following primary antibodies: rabbit anti–tankyrase1 762 (1 µg/ml) ; tankyrase1 465 (4 µg/ml) , rabbit anti-Flag (1 µg/ml) (Sigma Aldrich, F7425); rabbit anti-c-Myc (0.2 µg/ml) (Santa Cruz, sc-789); mouse anti-α-tubulin ascites (1:10,000) (Sigma Aldrich, T5768); rabbit anti-Hectd1 (1:1000) (Bethyl, A302-908A-T); rabbit anti-Naked1 (C30F10) (1:1000) (Cell Signaling, 2201); rabbit anti-HA (0.5 µg/ml) (Abcam, ab9110); rabbit anti-Naked2 (C67C4) (1:1000) (Cell Signaling, 2073); rabbit anti-VAMP8 (1:1000) (Bethyl A304-350A-T)or Abcam 76021 (1:1000); rabbit anti-DICER (1:1000) (Cell Signaling, 3363); rabbit anti-Notch2 (C-terminal) (D76A6)XP (1:1000) (Cell Signaling, 5732); rabbit anti-Notch2 (cleaved Val1697) (1:1000) (Sigma SAB4502022); rabbit anti-Notch1 (D6F11) (1:1000) (Cell Signaling 4380); rabbit anti-Notch3 (D11B8) (1:1000) (Cell Signaling 5276); rabbit anti-RNF146 (2.0 µg/ml)(Abcam ab106334); rabbit anti-Angiomotin (1:5000) (Bethyl, A303-305A-T-1); rabbit anti-Axin1 (C95H11) (1:1000) (Cell Signaling, 2074); mouse anti-HP1Δ (1:5000) (Millipore, MAB3450); rabbit anti-Chk2 (H-300) (0.2 µg/ml) (Santa Cruz, sc9064); or rabbit anti-GFP (2 µg/ml) (Abcam, ab290), followed by horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG (Amersham) (1:2500).

    Techniques: Binding Assay, Transfection, Immunoprecipitation

    E. fuscus Ig preferentially use lambda light chains . (Upper) Varying volumes of E. fuscus serum (expressed in μl) and swine IgG (expressed in μg) or (Lower) 0.03 μl of E. fuscus , BALB/c, or horse serum, as indicated, were fractionated using protein L-magnetic microbeads. The unbound ( λ ) and protein-L-binding ( κ ) material was reduced and then resolved using SDS-PAGE, followed by visualization using silver stain. Boxed areas represent individual IgH and IgL chains.

    Journal: Developmental and Comparative Immunology

    Article Title: Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody

    doi: 10.1016/j.dci.2016.06.024

    Figure Lengend Snippet: E. fuscus Ig preferentially use lambda light chains . (Upper) Varying volumes of E. fuscus serum (expressed in μl) and swine IgG (expressed in μg) or (Lower) 0.03 μl of E. fuscus , BALB/c, or horse serum, as indicated, were fractionated using protein L-magnetic microbeads. The unbound ( λ ) and protein-L-binding ( κ ) material was reduced and then resolved using SDS-PAGE, followed by visualization using silver stain. Boxed areas represent individual IgH and IgL chains.

    Article Snippet: For ELISAs, unlabeled and horseradish peroxidase (HRP)- conjugated goat anti-mouse Ig, goat anti-mouse IgG1, goat anti-mouse IgG2a, and goat anti-mouse IgM, as well as HRP-streptavidin were used (Southern Biotech).

    Techniques: Binding Assay, SDS Page, Silver Staining

    Serum Ig from different microchiropteran species is identified by Ab BT1-4F10 . Unreduced heart extracts from (1) E. fuscus , (2) M. lucifugus , (3) L. cinereus , (4) L. noctivagans , and (5) L. borealis bats were resolved by SDS-PAGE followed by immunoblotting with mAb BT1-4F10. All samples were assessed in the same experiment; shown are lanes cropped from samples of different volumes and exposures to permit normalized display of similar IgG band intensities. Similar sample volumes showed staining intensities of the order indicated by Table 2 . Data are representative of analysis of at least 5 individual bats per indicated species. All bats of a given species showed the same staining pattern.

    Journal: Developmental and Comparative Immunology

    Article Title: Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody

    doi: 10.1016/j.dci.2016.06.024

    Figure Lengend Snippet: Serum Ig from different microchiropteran species is identified by Ab BT1-4F10 . Unreduced heart extracts from (1) E. fuscus , (2) M. lucifugus , (3) L. cinereus , (4) L. noctivagans , and (5) L. borealis bats were resolved by SDS-PAGE followed by immunoblotting with mAb BT1-4F10. All samples were assessed in the same experiment; shown are lanes cropped from samples of different volumes and exposures to permit normalized display of similar IgG band intensities. Similar sample volumes showed staining intensities of the order indicated by Table 2 . Data are representative of analysis of at least 5 individual bats per indicated species. All bats of a given species showed the same staining pattern.

    Article Snippet: For ELISAs, unlabeled and horseradish peroxidase (HRP)- conjugated goat anti-mouse Ig, goat anti-mouse IgG1, goat anti-mouse IgG2a, and goat anti-mouse IgM, as well as HRP-streptavidin were used (Southern Biotech).

    Techniques: SDS Page, Staining

    mAb BT1-4F10 cross-reacts with bat and swine Ig light chain . A 50% SAS precipitate of E. fuscus serum or purified swine IgG were resolved by SDS-PAGE and immunoblotted with mAB 4F10. Reduced and non-reduced samples were run on the same gel; lanes were cropped to remove irrelevant lanes.

    Journal: Developmental and Comparative Immunology

    Article Title: Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody

    doi: 10.1016/j.dci.2016.06.024

    Figure Lengend Snippet: mAb BT1-4F10 cross-reacts with bat and swine Ig light chain . A 50% SAS precipitate of E. fuscus serum or purified swine IgG were resolved by SDS-PAGE and immunoblotted with mAB 4F10. Reduced and non-reduced samples were run on the same gel; lanes were cropped to remove irrelevant lanes.

    Article Snippet: For ELISAs, unlabeled and horseradish peroxidase (HRP)- conjugated goat anti-mouse Ig, goat anti-mouse IgG1, goat anti-mouse IgG2a, and goat anti-mouse IgM, as well as HRP-streptavidin were used (Southern Biotech).

    Techniques: Purification, SDS Page