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  • 99
    Vector Laboratories biotinylated goat anti rabbit igg
    Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary <t>biotinylated</t> goat anti-rabbit <t>IgG</t> (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.
    Biotinylated Goat Anti Rabbit Igg, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 9104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    LI-COR irdye 800cw goat anti rabbit igg h l
    All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary <t>IRDye</t> <t>800CW-conjugated</t> goat (polyclonal) anti-rabbit <t>IgG</t> (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.
    Irdye 800cw Goat Anti Rabbit Igg H L, supplied by LI-COR, used in various techniques. Bioz Stars score: 99/100, based on 3552 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher goat anti rabbit igg h l cross adsorbed secondary antibody
    All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary <t>IRDye</t> <t>800CW-conjugated</t> goat (polyclonal) anti-rabbit <t>IgG</t> (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.
    Goat Anti Rabbit Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 14192 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 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher goat anti rabbit 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
    Goat Anti Rabbit Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher goat 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
    Goat Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti rabbit igg whole molecule peroxidase antibody
    Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by <t>immunoblotting</t> (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit <t>IgG</t> (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P
    Anti Rabbit Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam goat anti rabbit igg h l hrp
    F-ATPase/VGCC protein complex verification. a. 1D immunoblot analysis of the VGCC α2δ-1 subunit in neutrophils. The VGCC α2δ-1 subunit-containing protein complexes in a NativePAGE Novex 4–16% Bis-Tris gel were detected at approximately 750 kDa via immunoblotting, the same position as the F-ATPase F1 β-subunit. b. 2D immunoblot analysis of the F-ATPase and VGCC protein complex. Plasma membrane lysed by 2% digitonin was loaded and run on a BN-PAGE gel. One lane (Sample) was cut and equilibrated in SDS loading buffer, followed by fixation on a SDS-PAGE gel for 2D electrophoretic analysis. Two separate immunoblot analyses with a primary antibody against the VGCC α2δ-1 subunit or the F-ATPase F1 β-subunit were performed. The F-ATPase F1 β-subunit appears vertically below the VGCC α2δ-1 subunit in the same PVDF membrane. c. Immunofluorescence colocalization analysis of F-ATPase and VGCC. Representative images show that the VGCC α2δ-1 subunit and F-ATPase F1 β-subunit colocalized on the plasma membrane of neutrophils. d. Immunoprecipitation of VGCC α2δ-1 subunit and F-ATPase from plasma membrane protein in human neutrophils. Samples were immunoprecipitated with a mouse monoclonal antibody against α2 of the VGCC α2δ-1 subunit. F-ATPase was detected with a mouse monoclonal antibody against the F-ATPase F1 α-subunit or β-subunit and an <t>HRP-conjugated</t> goat anti-mouse <t>IgG</t> light chain specifically bound antibody. IgG control experiment did not detect F-ATPase
    Goat Anti Rabbit Igg H L Hrp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2991 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher alexa fluor 488 goat anti rabbit igg
    Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with <t>Alexa</t> <t>Fluor</t> 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p
    Alexa Fluor 488 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 7939 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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    94
    Thermo Fisher goat anti rabbit igg
    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells <t>(RBCs)</t> to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. <t>IgG1,</t> immunoglobulin G1.
    Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10674 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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    91
    Santa Cruz Biotechnology goat anti rabbit igg horseradish peroxidase
    S. aureus EV-associated BlaZ was resistant to proteinase K treatment. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ and EVs, which were digested with proteinase K, detected with lab-made <t>polyclonal</t> anti-BlaZ antibodies. SbI indicates the band of <t>IgG-binding</t> protein SbI. Molecular weight standards are indicated on the left (kDa). (B) Effect of intact and proteinase K (PK)-treated S. aureus ATCC 14458 soluble BlaZ and EVs on the growth of ampicillin-susceptible E. coli DH5α in LB-ampicillin (100 μg/ml) medium. ***, P
    Goat Anti Rabbit Igg Horseradish Peroxidase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher donkey anti goat igg h l cross adsorbed secondary antibody
    S. aureus EV-associated BlaZ was resistant to proteinase K treatment. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ and EVs, which were digested with proteinase K, detected with lab-made <t>polyclonal</t> anti-BlaZ antibodies. SbI indicates the band of <t>IgG-binding</t> protein SbI. Molecular weight standards are indicated on the left (kDa). (B) Effect of intact and proteinase K (PK)-treated S. aureus ATCC 14458 soluble BlaZ and EVs on the growth of ampicillin-susceptible E. coli DH5α in LB-ampicillin (100 μg/ml) medium. ***, P
    Donkey Anti Goat Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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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    99
    Jackson Immuno peroxidase affinipure goat anti rabbit igg
    Dose-response curves of Ca 2+ uptake in A549 cells and Western blot panel showing CHRNA7 gene silencing. Cells were treated with various concentrations of APS8 ( A ; 10, 5, 2.5, 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01 µM) or ACh ( B ; 100, 40, 20, 10, 5, 2.5, 1, 0.5, 0.1, 0.02 µM) or preincubated (20 min, RT) with various concentrations of APS8 and then treated with 100 µM ACh ( C ). The A549 cells were silenced with CHRNA7-targeted or nontargeting (negative control) siRNAs. Data are relative fluorescence units (RFU), as maximum response minus minimum response, divided by the minimum response. The curves and their 95% confidence intervals were obtained by fitting the data with nonlinear regression function (GraphPad Prism). Recombinant human α7 nAChR (#H00001139-P01, Abnova; Lane 1) and lysates of A549 cells treated with nontargeting siRNA (Lane 2) or siRNA targeting CHRNA7 (Lanes 3 and 4) were separated by SDS-PAGE followed by Western blotting ( D ) using rabbit antibodies against human α7 nAChR (1:500; #HPA029422, Sigma-Aldrich) and Peroxidase <t>AffiniPure</t> Goat Anti-Rabbit <t>IgG</t> (1:10,000; #111-035-003, Jackson ImmunoResearch Europe Ltd).
    Peroxidase Affinipure Goat Anti Rabbit Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 4084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher goat anti rat igg h l cross adsorbed secondary antibody
    Dose-response curves of Ca 2+ uptake in A549 cells and Western blot panel showing CHRNA7 gene silencing. Cells were treated with various concentrations of APS8 ( A ; 10, 5, 2.5, 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01 µM) or ACh ( B ; 100, 40, 20, 10, 5, 2.5, 1, 0.5, 0.1, 0.02 µM) or preincubated (20 min, RT) with various concentrations of APS8 and then treated with 100 µM ACh ( C ). The A549 cells were silenced with CHRNA7-targeted or nontargeting (negative control) siRNAs. Data are relative fluorescence units (RFU), as maximum response minus minimum response, divided by the minimum response. The curves and their 95% confidence intervals were obtained by fitting the data with nonlinear regression function (GraphPad Prism). Recombinant human α7 nAChR (#H00001139-P01, Abnova; Lane 1) and lysates of A549 cells treated with nontargeting siRNA (Lane 2) or siRNA targeting CHRNA7 (Lanes 3 and 4) were separated by SDS-PAGE followed by Western blotting ( D ) using rabbit antibodies against human α7 nAChR (1:500; #HPA029422, Sigma-Aldrich) and Peroxidase <t>AffiniPure</t> Goat Anti-Rabbit <t>IgG</t> (1:10,000; #111-035-003, Jackson ImmunoResearch Europe Ltd).
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    Millipore igg4
    SmLy6A and SmLy6B are differentially recognised in a cohort of S . mansoni infected males from a Ugandan fishing community. Recombinant SmLy6A and SmLy6B were used in ELISA to measure antigen-specific <t>IgG</t> 1 (SmLy6A—Panel A, SmLy6B—Panel D), IgG 4 (SmLy6A—Panel B and SmLy6B—Panel E) and IgE (SmLy6A—Panel C and SmLy6B—Panel F) responses in the plasma of 216 males infected with S . mansoni prior to praziquantel treatment. The cohort is divided into 5 age groups, 7–9 (n = 36), 10–13 (n = 37), 14–23 (n = 41), 24–32 (n = 42) and 33+ (n = 60) and the antibody levels of each group is presented as box plots.
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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 .
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    Thermo Fisher alexa fluor 488 conjugated goat anti rabbit igg
    Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with <t>Alexa</t> Fluor 488-conjugated anti-rabbit
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    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.
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    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.
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    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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    Santa Cruz Biotechnology goat anti rabbit igg
    Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with <t>IgG</t> (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP + T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the <t>immunoblots.</t> (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E . Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.
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    Image Search Results


    Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV

    doi: 10.3390/ijms21072353

    Figure Lengend Snippet: Distribution of avian influenza virus NP in selected organs of inoculated birds. Distribution of influenza NP in brain ( A , E , I , M ), lung ( B , F , J , N ), spleen ( C , G , K , O ) and heart ( D , H , L , P ) of inoculated chickens of selected viruses at 4 dpi (except for H5N1_H4_T 327 K) as detected by immunohistochemistry using primary polyclonal rabbit anti-NP A/FPV/Rostock/34 antibody (1:750) and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200). 3-amino-9-ethyl-carbazol (red-brown); hematoxylin counterstain (blue); Nomarski contrast; bars A,B,D,E,F,H,I,J,L,M,N,P = 20 µm. Bars C,G,K,O = 50 µm.

    Article Snippet: Following sections were used for immunohistochemistry using the avidin–biotin–peroxidase complex method (Vector Laboratories Burlingame, CA, USA) with a primary polyclonal rabbit anti-NP antibody (1:750), and a secondary biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) antibody (1:200) as described [ , ].

    Techniques: Immunohistochemistry, Plasmid Preparation

    Human BACE1 immunohistochemical localization Brains from 2-month-old BACE1 transgenic ( B, D, F ) and nontransgenic ( A, C, E ) animals were cryoprotected in 30% sucrose after perfusion with 4% paraformaldehyde. Sagittal sections were analyzed by staining with polyclonal antibody B279 followed by staining with biotinylated anti-goat secondary antibody ( A - F ) or Alexa-488-conjugated secondary antibody ( G and H ). Positive immunoreactivity is present in the olfactory bulb ( A and B ), hippocampus ( C and D ), and frontal cortex ( E and F ). Scale bar in F and H = 200 μ m.

    Journal: The Journal of biological chemistry

    Article Title: Altered Amyloid-β Metabolism and Deposition in Genomic-based β-Secretase Transgenic Mice *

    doi: 10.1074/jbc.M409680200

    Figure Lengend Snippet: Human BACE1 immunohistochemical localization Brains from 2-month-old BACE1 transgenic ( B, D, F ) and nontransgenic ( A, C, E ) animals were cryoprotected in 30% sucrose after perfusion with 4% paraformaldehyde. Sagittal sections were analyzed by staining with polyclonal antibody B279 followed by staining with biotinylated anti-goat secondary antibody ( A - F ) or Alexa-488-conjugated secondary antibody ( G and H ). Positive immunoreactivity is present in the olfactory bulb ( A and B ), hippocampus ( C and D ), and frontal cortex ( E and F ). Scale bar in F and H = 200 μ m.

    Article Snippet: All sections were incubated in biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA).

    Techniques: Immunohistochemistry, Transgenic Assay, Staining

    All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary IRDye 800CW-conjugated goat (polyclonal) anti-rabbit IgG (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.

    Journal: mBio

    Article Title: Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

    doi: 10.1128/mBio.02285-14

    Figure Lengend Snippet: All of the gp42 linker mutants are expressed on the cell surface and are cleaved and secreted. CHO-KI cells were transiently transfected in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were transfected overnight with 4 µg each vector alone (pCAGGS), wild-type gp42, d37-41gp42, d36FLAGgp42, d37-41(I27-1), d37-41(I27-1,2), and d31-44 (I27-1,2,3,4), followed by a medium change to Ham’s F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Forty-eight hours posttransfection, the supernatants were collected, and the cells were lysed in Triton X-100 lysis buffer containing protease inhibitors. (A and B) Cell lysates (A) and culture supernatants (B) were analyzed by 12% SDS-PAGE and Western blotting with polyclonal anti-gp42 antibody (PB114; R. Longnecker) and secondary IRDye 800CW-conjugated goat (polyclonal) anti-rabbit IgG (H+L) (catalog no. 926-32211; Li-Cor Biosciences) as previously described. Blots were washed and analyzed with Li-Cor Biosciences Odyssey infrared imaging studio software. The positions of molecular size markers (in kilodaltons) are indicated to the left of the blots. Although the blots in panels A and B were from the same gel, intervening lanes that were not applicable to the experiment shown were removed.

    Article Snippet: The blots were washed, and IRDye 800CW-conjugated goat (polyclonal) anti-rabbit IgG (H+L) (catalog no. 926-32211; Li-Cor Biosciences) diluted 1:10,000 in blocking solution was applied for 1 h at room temperature with an aluminum foil cover.

    Techniques: Transfection, Plasmid Preparation, Lysis, SDS Page, Western Blot, Imaging, Software

    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

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms12526

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

    Article Snippet: Secondary antibodies used in immunoblotting are goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (Sigma, A2554, 1:5,000), goat anti-rabbit IgG HRP-conjugated antibody (Sigma, A0545, 1:5,000) and rabbit anti-goat IgG HRP-conjugated antibody (Sigma, A5420, 1:5,000).

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

    F-ATPase/VGCC protein complex verification. a. 1D immunoblot analysis of the VGCC α2δ-1 subunit in neutrophils. The VGCC α2δ-1 subunit-containing protein complexes in a NativePAGE Novex 4–16% Bis-Tris gel were detected at approximately 750 kDa via immunoblotting, the same position as the F-ATPase F1 β-subunit. b. 2D immunoblot analysis of the F-ATPase and VGCC protein complex. Plasma membrane lysed by 2% digitonin was loaded and run on a BN-PAGE gel. One lane (Sample) was cut and equilibrated in SDS loading buffer, followed by fixation on a SDS-PAGE gel for 2D electrophoretic analysis. Two separate immunoblot analyses with a primary antibody against the VGCC α2δ-1 subunit or the F-ATPase F1 β-subunit were performed. The F-ATPase F1 β-subunit appears vertically below the VGCC α2δ-1 subunit in the same PVDF membrane. c. Immunofluorescence colocalization analysis of F-ATPase and VGCC. Representative images show that the VGCC α2δ-1 subunit and F-ATPase F1 β-subunit colocalized on the plasma membrane of neutrophils. d. Immunoprecipitation of VGCC α2δ-1 subunit and F-ATPase from plasma membrane protein in human neutrophils. Samples were immunoprecipitated with a mouse monoclonal antibody against α2 of the VGCC α2δ-1 subunit. F-ATPase was detected with a mouse monoclonal antibody against the F-ATPase F1 α-subunit or β-subunit and an HRP-conjugated goat anti-mouse IgG light chain specifically bound antibody. IgG control experiment did not detect F-ATPase

    Journal: Cell Communication and Signaling : CCS

    Article Title: F0F1 ATP synthase regulates extracellular calcium influx in human neutrophils by interacting with Cav2.3 and modulates neutrophil accumulation in the lipopolysaccharide-challenged lung

    doi: 10.1186/s12964-020-0515-3

    Figure Lengend Snippet: F-ATPase/VGCC protein complex verification. a. 1D immunoblot analysis of the VGCC α2δ-1 subunit in neutrophils. The VGCC α2δ-1 subunit-containing protein complexes in a NativePAGE Novex 4–16% Bis-Tris gel were detected at approximately 750 kDa via immunoblotting, the same position as the F-ATPase F1 β-subunit. b. 2D immunoblot analysis of the F-ATPase and VGCC protein complex. Plasma membrane lysed by 2% digitonin was loaded and run on a BN-PAGE gel. One lane (Sample) was cut and equilibrated in SDS loading buffer, followed by fixation on a SDS-PAGE gel for 2D electrophoretic analysis. Two separate immunoblot analyses with a primary antibody against the VGCC α2δ-1 subunit or the F-ATPase F1 β-subunit were performed. The F-ATPase F1 β-subunit appears vertically below the VGCC α2δ-1 subunit in the same PVDF membrane. c. Immunofluorescence colocalization analysis of F-ATPase and VGCC. Representative images show that the VGCC α2δ-1 subunit and F-ATPase F1 β-subunit colocalized on the plasma membrane of neutrophils. d. Immunoprecipitation of VGCC α2δ-1 subunit and F-ATPase from plasma membrane protein in human neutrophils. Samples were immunoprecipitated with a mouse monoclonal antibody against α2 of the VGCC α2δ-1 subunit. F-ATPase was detected with a mouse monoclonal antibody against the F-ATPase F1 α-subunit or β-subunit and an HRP-conjugated goat anti-mouse IgG light chain specifically bound antibody. IgG control experiment did not detect F-ATPase

    Article Snippet: Voltage-gated calcium channel (VGCC) α2δ-1 was detected with a rabbit polyclonal antibody against the VGCC α2δ-1 subunit (1:200, C5105, Sigma-Aldrich, St. Louis, MO, USA) and an HRP-conjugated goat anti-rabbit IgG H & L (1:2000, ab6721, Abcam, Cambridge, UK).

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Immunofluorescence, Immunoprecipitation

    Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p

    Journal: Arthritis Research & Therapy

    Article Title: Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

    doi: 10.1186/s13075-018-1693-x

    Figure Lengend Snippet: Colocalization of rhodamine-labeled recombinant human proteoglycan-4 (rhPRG4) (red) and isotype control (IC), CD44 (probed using anti-CD44), toll-like receptor 2 (TLR2) (probed using anti-TLR2) or toll-like receptor 4 (TLR4) (probed using anti-TLR4) in peritoneal Prg4 −/− murine macrophages. Cells were incubated with rhodamine-rhPRG4 for 2 h followed by cell fixation and permeabilization. Following receptor probing, cells were incubated with Alexa Fluor 488 conjugated secondary antibody (green) and counterstained with DAPI (blue). Arrows point to co-localization of rhPRG4 with respective receptors. Quantitative colocalization analysis was performed using Pearson’s Correlation Coefficient and a cutoff of r 2 > 0.5 was used to indicate positive colocalization. The percentage of cells with positive colocalization was determined and at least 100 cells were examined for each treatment condition. Data represent the mean ± S.D. of three independent experiments. Median colocalization images are presented. * p

    Article Snippet: Cells were then washed with PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific) at 1:200 dilution for 1 h at room temperature.

    Techniques: Labeling, Recombinant, Incubation

    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

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.

    Journal: The Journal of Infectious Diseases

    Article Title: Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA

    doi: 10.1093/infdis/jiy467

    Figure Lengend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.

    Article Snippet: Rabbit anti-VAR2CSA or normal rabbit serum was preabsorbed on uninfected RBCs, incubated with infected RBCs at a ratio of 1:40, and detected with AlexaFluor 647–conjugated goat anti-rabbit IgG (dilution, 1:500; Life Technologies).

    Techniques: Binding Assay, Infection, Inhibition, Incubation

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.

    Journal: The Journal of Infectious Diseases

    Article Title: Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA

    doi: 10.1093/infdis/jiy467

    Figure Lengend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.

    Article Snippet: Rabbit anti-VAR2CSA or normal rabbit serum was preabsorbed on uninfected RBCs, incubated with infected RBCs at a ratio of 1:40, and detected with AlexaFluor 647–conjugated goat anti-rabbit IgG (dilution, 1:500; Life Technologies).

    Techniques: Binding Assay, Infection, Flow Cytometry, Cytometry, Expressing, Staining

    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

    S. aureus EV-associated BlaZ was resistant to proteinase K treatment. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ and EVs, which were digested with proteinase K, detected with lab-made polyclonal anti-BlaZ antibodies. SbI indicates the band of IgG-binding protein SbI. Molecular weight standards are indicated on the left (kDa). (B) Effect of intact and proteinase K (PK)-treated S. aureus ATCC 14458 soluble BlaZ and EVs on the growth of ampicillin-susceptible E. coli DH5α in LB-ampicillin (100 μg/ml) medium. ***, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Staphylococcus aureus Extracellular Vesicles Carry Biologically Active ?-Lactamase

    doi: 10.1128/AAC.00522-12

    Figure Lengend Snippet: S. aureus EV-associated BlaZ was resistant to proteinase K treatment. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ and EVs, which were digested with proteinase K, detected with lab-made polyclonal anti-BlaZ antibodies. SbI indicates the band of IgG-binding protein SbI. Molecular weight standards are indicated on the left (kDa). (B) Effect of intact and proteinase K (PK)-treated S. aureus ATCC 14458 soluble BlaZ and EVs on the growth of ampicillin-susceptible E. coli DH5α in LB-ampicillin (100 μg/ml) medium. ***, P

    Article Snippet: The blocked membrane was incubated with the lab-made polyclonal anti-BlaZ antibody and then incubated with goat anti-rabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Western Blot, Purification, Binding Assay, Molecular Weight

    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

    Dose-response curves of Ca 2+ uptake in A549 cells and Western blot panel showing CHRNA7 gene silencing. Cells were treated with various concentrations of APS8 ( A ; 10, 5, 2.5, 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01 µM) or ACh ( B ; 100, 40, 20, 10, 5, 2.5, 1, 0.5, 0.1, 0.02 µM) or preincubated (20 min, RT) with various concentrations of APS8 and then treated with 100 µM ACh ( C ). The A549 cells were silenced with CHRNA7-targeted or nontargeting (negative control) siRNAs. Data are relative fluorescence units (RFU), as maximum response minus minimum response, divided by the minimum response. The curves and their 95% confidence intervals were obtained by fitting the data with nonlinear regression function (GraphPad Prism). Recombinant human α7 nAChR (#H00001139-P01, Abnova; Lane 1) and lysates of A549 cells treated with nontargeting siRNA (Lane 2) or siRNA targeting CHRNA7 (Lanes 3 and 4) were separated by SDS-PAGE followed by Western blotting ( D ) using rabbit antibodies against human α7 nAChR (1:500; #HPA029422, Sigma-Aldrich) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (1:10,000; #111-035-003, Jackson ImmunoResearch Europe Ltd).

    Journal: Marine Drugs

    Article Title: APS8 Delays Tumor Growth in Mice by Inducing Apoptosis of Lung Adenocarcinoma Cells Expressing High Number of α7 Nicotinic Receptors

    doi: 10.3390/md16100367

    Figure Lengend Snippet: Dose-response curves of Ca 2+ uptake in A549 cells and Western blot panel showing CHRNA7 gene silencing. Cells were treated with various concentrations of APS8 ( A ; 10, 5, 2.5, 1, 0.75, 0.5, 0.25, 0.1, 0.05, 0.01 µM) or ACh ( B ; 100, 40, 20, 10, 5, 2.5, 1, 0.5, 0.1, 0.02 µM) or preincubated (20 min, RT) with various concentrations of APS8 and then treated with 100 µM ACh ( C ). The A549 cells were silenced with CHRNA7-targeted or nontargeting (negative control) siRNAs. Data are relative fluorescence units (RFU), as maximum response minus minimum response, divided by the minimum response. The curves and their 95% confidence intervals were obtained by fitting the data with nonlinear regression function (GraphPad Prism). Recombinant human α7 nAChR (#H00001139-P01, Abnova; Lane 1) and lysates of A549 cells treated with nontargeting siRNA (Lane 2) or siRNA targeting CHRNA7 (Lanes 3 and 4) were separated by SDS-PAGE followed by Western blotting ( D ) using rabbit antibodies against human α7 nAChR (1:500; #HPA029422, Sigma-Aldrich) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (1:10,000; #111-035-003, Jackson ImmunoResearch Europe Ltd).

    Article Snippet: This was followed by Western blotting with rabbit antibodies against human α7 nAChR (1:500; #HPA029422, Sigma-Aldrich) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (1:10,000; #111-035-003, Jackson ImmunoResearch Europe Ltd, Ely, Cambridgeshire, UK).

    Techniques: Western Blot, Negative Control, Fluorescence, Recombinant, SDS Page

    SmLy6A and SmLy6B are differentially recognised in a cohort of S . mansoni infected males from a Ugandan fishing community. Recombinant SmLy6A and SmLy6B were used in ELISA to measure antigen-specific IgG 1 (SmLy6A—Panel A, SmLy6B—Panel D), IgG 4 (SmLy6A—Panel B and SmLy6B—Panel E) and IgE (SmLy6A—Panel C and SmLy6B—Panel F) responses in the plasma of 216 males infected with S . mansoni prior to praziquantel treatment. The cohort is divided into 5 age groups, 7–9 (n = 36), 10–13 (n = 37), 14–23 (n = 41), 24–32 (n = 42) and 33+ (n = 60) and the antibody levels of each group is presented as box plots.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment

    doi: 10.1371/journal.pntd.0003920

    Figure Lengend Snippet: SmLy6A and SmLy6B are differentially recognised in a cohort of S . mansoni infected males from a Ugandan fishing community. Recombinant SmLy6A and SmLy6B were used in ELISA to measure antigen-specific IgG 1 (SmLy6A—Panel A, SmLy6B—Panel D), IgG 4 (SmLy6A—Panel B and SmLy6B—Panel E) and IgE (SmLy6A—Panel C and SmLy6B—Panel F) responses in the plasma of 216 males infected with S . mansoni prior to praziquantel treatment. The cohort is divided into 5 age groups, 7–9 (n = 36), 10–13 (n = 37), 14–23 (n = 41), 24–32 (n = 42) and 33+ (n = 60) and the antibody levels of each group is presented as box plots.

    Article Snippet: Standard curves were generated by control immunoglobulins including IgE (Calbiochem), IgG1 (Sigma) or IgG4 (Sigma) as appropriate.

    Techniques: Infection, Recombinant, Enzyme-linked Immunosorbent Assay

    Praziquantel treatment does not alter SmLy6A and SmLy6B IgG 1 age distribution profiles, but does decrease antigen-specific IgG 1 reactivity. SmLy6A-, SmLy6B- and SmTAL1-specific IgG 1 were measured before and 9 weeks after praziquantel treatment in a cohort of infected males (n = 216). A) Age–associated profiles of SmLy6A-, B) SmLy6B-, and C) SmTAL1-specific IgG 1 responses pre- and post-praziquantel treatment. (D) Effect of praziquantel treatment on human IgG 1 responses to SmLy6A, (E) SmLy6B and (F) SmTAL1. IgG 1 levels for those individuals producing a detectable response ( > mean+3xSD uninfected controls) to SmLy6A (n = 16), SmLy6B (n = 50) or SmTAL1 (n = 26) before treatment are shown and the median value is represented by a horizontal bar. P -values were calculated using a Wilcoxon signed-rank test comparing pre-treatment and post-treatment antibody levels.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment

    doi: 10.1371/journal.pntd.0003920

    Figure Lengend Snippet: Praziquantel treatment does not alter SmLy6A and SmLy6B IgG 1 age distribution profiles, but does decrease antigen-specific IgG 1 reactivity. SmLy6A-, SmLy6B- and SmTAL1-specific IgG 1 were measured before and 9 weeks after praziquantel treatment in a cohort of infected males (n = 216). A) Age–associated profiles of SmLy6A-, B) SmLy6B-, and C) SmTAL1-specific IgG 1 responses pre- and post-praziquantel treatment. (D) Effect of praziquantel treatment on human IgG 1 responses to SmLy6A, (E) SmLy6B and (F) SmTAL1. IgG 1 levels for those individuals producing a detectable response ( > mean+3xSD uninfected controls) to SmLy6A (n = 16), SmLy6B (n = 50) or SmTAL1 (n = 26) before treatment are shown and the median value is represented by a horizontal bar. P -values were calculated using a Wilcoxon signed-rank test comparing pre-treatment and post-treatment antibody levels.

    Article Snippet: Standard curves were generated by control immunoglobulins including IgE (Calbiochem), IgG1 (Sigma) or IgG4 (Sigma) as appropriate.

    Techniques: 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

    Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with Alexa Fluor 488-conjugated anti-rabbit

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Multicopper oxidase-1 is a ferroxidase essential for iron homeostasis in Drosophila melanogaster

    doi: 10.1073/pnas.1208703109

    Figure Lengend Snippet: Immunolocalization of MCO1. Cryosections of larval ( A ) and adult ( B ) midguts and unsectioned larval ( C ) and adult ( D ) Malpighian tubules were immunostained with MCO1 antiserum. Anti-MCO1 antibodies were detected with Alexa Fluor 488-conjugated anti-rabbit

    Article Snippet: Briefly, dissected guts from wandering larvae or adult females were fixed in 4% (wt/vol) paraformaldehyde in PBS solution for 1 h, sections were incubated for 2 h with partially purified MCO1 antiserum or preimmune serum, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) was used as the secondary antibody.

    Techniques:

    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

    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

    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

    Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP + T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E . Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.

    Journal: The Journal of Cell Biology

    Article Title: The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1

    doi: 10.1083/jcb.200801121

    Figure Lengend Snippet: Affect of Abl suppression on CrkL–C3G membrane localization and activation. (A) Jurkat T cells transfected with control, WAVE2, or Abl EGFP suppression vectors were stimulated with IgG (column 1) or OKT3-coated beads (columns 2–4) and imaged for CrkL (aqua) and F-actin (red) recruitment. The percentage of EGFP + T cell–bead conjugates showing localization of CrkL is indicated and was performed as described in Materials and methods. Representative images are shown. Bar, 5 μm. (B) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking and cytosolic/membrane fractions, were prepared and immunoblotted with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots. (C) Purified Abl and Fyn were incubated with the indicated GST fusion proteins in kinase buffer, and tyrosine phosphorylation was detected by anti-Tyr immunoblotting. Input levels of GST were detected by anti-GST immunoblotting. Numbers on the left are arbitrary units based on densitometric analysis of the immunoblots. (D) Jurkat T cells were transfected with the indicated suppression vectors, stimulated by anti-CD3 cross-linking, and C3G was precipitated as described in Fig. 5 E . Proteins were detected by immunoblotting with the indicated antibodies. Numbers below blots are arbitrary units based on densitometric analysis of the immunoblots.

    Article Snippet: For immunoblots, antibodies were detected using goat anti–mouse IgG or goat anti–rabbit IgG coupled to HRP (Santa Cruz Biotechnology, Inc.) and SuperSignal Enhanced Chemiluminescence (Thermo Fisher Scientific).

    Techniques: Activation Assay, Transfection, Western Blot, Purification, Incubation