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  • 95
    Thermo Fisher anti rabbit igg
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 7812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti ctcf
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Anti Ctcf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Nordic-Mubio rabbit igg
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Rabbit Igg, supplied by Nordic-Mubio, used in various techniques. Bioz Stars score: 91/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Rockland Immunochemicals rabbit igg4 irdye800
    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. <t>Biotinylated</t> anti-rabbit <t>IgG</t> followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.
    Rabbit Igg4 Irdye800, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rabbit igg
    STAiR1 – a continuous specifically expressed transcript. (A) INA6 cells were restimulated with IL-6 as described in Figure 3 A and chromatin immunoprecipitated (ChIP-ed) for tri-methylated H3K4 and H3K36, respectively. Enrichment compared to an <t>IgG</t> isotype control was assessed by quantitative real-time PCR using primer sets P1, P3, P5 and P6. The location of respective amplicons is shown in Figure 3 A. Strong enrichment for <t>H3K4me3</t> is observed only within P1, indicating an active promoter region. H3K36me3 shows strong enrichment throughout the STAiR1 transcript. (B) Expression z -score aggregated over STAiR1 expressed after 1 h (STAiR1 short, chr18:41,591,020-41,720,348) or the entire annotated STAiR1 transcript (STAiR1 long). (C) INA6 cells were restimulated with IL-6 as described and induction of STAiR1 was detected using qRT-PCR with primer sets P1 to P6, as shown in Figure 3 A, and using GAPDH for normalization. This expression time course is consistent with the time-dependent elongation of STAiR1 observed in the tiling array data shown in Figure 3 A. (D) Expression of macroRNAs in different tissues, as detected by reverse transcriptase PCR, using GAPDH as a normalization control. Tissue specificity varies strongly between different macroRNAs. STAiR, STAT3-induced RNA; STAT3, signal transducer and activator of transcription-3.
    Rabbit Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies rabbit igg
    Autoantibodies and increased B cells and plasmablasts in CD11c-Flip-KO mice. ( a ) Quantification of serum <t>IgG</t> subclasses by <t>ELISA</t> (μg ml −1 ) in the CD11c-Flip-KO (KO) and control mice. ( b ) ELISA quantification of serum RF, ANA and antiCCP (lower panel). Values are presented as OD 450 × serum dilution for RF and anti-CCP and units for ANA. ( c , d ) Immunoblotting of ankle ( c ) and kidney ( d ) homogenates from Rag −/− mice employing individual serum randomly selected from control and CD11c-Flip-KO mice (≥20 weeks) and a duplicated tissue blot stained with Coomassie blue R250. The arrows identify protein bands common between individual CD11c-Flip-KO but not in control mice. The blots are representative of three independent experiments. ( e , f ) Antibodies identified by autoantigen array. ( e ). Heatmap of IgG antibodies to joint-related antigens, generated as the signal-to-noise ratio (SNR) for each antigen (controls n =7, CD11c-Flip-KO mice ≥20 weeks with arthritis, n =8). ( f ) Three autoantibodies in the array were significantly increased following the Bonferroni correction in the CD11c-Flip-KO mice. ( g , h ) pLNs were examined for B cells ( n =12–15 per group) and plasmablasts ( n =6 per group) from mice ≥20 weeks. B cells were defined as CD19 + B220 + and plasmablasts as CD19 + B220 + CD138 + in the CD64 − CD11b − population. ( i ) Correlation (Pearson's) between the number of pLN plasmablasts and the arthritis clinical score for CD11c-Flip-KO mice just before killing. The values represent the mean±1 s.e. (* P
    Rabbit Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore rabbit igg antihuman igg
    Autoantibodies and increased B cells and plasmablasts in CD11c-Flip-KO mice. ( a ) Quantification of serum <t>IgG</t> subclasses by <t>ELISA</t> (μg ml −1 ) in the CD11c-Flip-KO (KO) and control mice. ( b ) ELISA quantification of serum RF, ANA and antiCCP (lower panel). Values are presented as OD 450 × serum dilution for RF and anti-CCP and units for ANA. ( c , d ) Immunoblotting of ankle ( c ) and kidney ( d ) homogenates from Rag −/− mice employing individual serum randomly selected from control and CD11c-Flip-KO mice (≥20 weeks) and a duplicated tissue blot stained with Coomassie blue R250. The arrows identify protein bands common between individual CD11c-Flip-KO but not in control mice. The blots are representative of three independent experiments. ( e , f ) Antibodies identified by autoantigen array. ( e ). Heatmap of IgG antibodies to joint-related antigens, generated as the signal-to-noise ratio (SNR) for each antigen (controls n =7, CD11c-Flip-KO mice ≥20 weeks with arthritis, n =8). ( f ) Three autoantibodies in the array were significantly increased following the Bonferroni correction in the CD11c-Flip-KO mice. ( g , h ) pLNs were examined for B cells ( n =12–15 per group) and plasmablasts ( n =6 per group) from mice ≥20 weeks. B cells were defined as CD19 + B220 + and plasmablasts as CD19 + B220 + CD138 + in the CD64 − CD11b − population. ( i ) Correlation (Pearson's) between the number of pLN plasmablasts and the arthritis clinical score for CD11c-Flip-KO mice just before killing. The values represent the mean±1 s.e. (* P
    Rabbit Igg Antihuman Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc rabbit igg
    14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells. a) Cell lysates from M17 cells were immunoprecipitated with a <t>polyclonal</t> rabbit antibody against Bax or rabbit <t>IgG,</t> and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. b) Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).
    Rabbit Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 3222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sino Biological rabbit igg
    14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells. a) Cell lysates from M17 cells were immunoprecipitated with a <t>polyclonal</t> rabbit antibody against Bax or rabbit <t>IgG,</t> and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. b) Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).
    Rabbit Igg, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore non specific rabbit igg1
    14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells. a) Cell lysates from M17 cells were immunoprecipitated with a <t>polyclonal</t> rabbit antibody against Bax or rabbit <t>IgG,</t> and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. b) Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).
    Non Specific Rabbit Igg1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc normal rabbit igg
    Expression of <t>ETS2,</t> DLX3, and POU5F1 in JAr cell lines. A, Coimmunoprecipitation analysis to determine any physical interaction between ETS2 and DLX3. Immunoprecipitation was performed with antibody to DLX3 and Western analysis with affinity-purified ETS2 antibody. Lane 1 (input), Thirty micrograms of control cell lysate; lane 2 (anti-ETS2), ETS2-DLX3 complexes from control cell lysate; lane 3 (control <t>IgG),</t> immunocomplexes collected after the addition of a nonspecific IgG; lane 4 (anti-ETS2), extracts from cells transfected with both ETS2 and DLX3 expression plasmids (3 μg plasmid DNA/dish). B, Detection of ETS2 and DLX3 on Western blots of lysates from the S1 and S4 lines (which stably express POU5F1), from the C1 and C2 lines (which had been transfected with an empty vector at the same time that the S1 and S2 lines had been generated), and a control JAr cell line (JAr). The loading control was ACTB ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (60 μg) in each lane. C, POU5F1 detection in the S1, S4, C1, and C2 cell lines, with ACTB as a loading control in the lower panel . Molecular weight markers (M r × 10 −3 ) are shown on the left .
    Normal Rabbit Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    SouthernBiotech rabbit igg1 control
    Expression of <t>ETS2,</t> DLX3, and POU5F1 in JAr cell lines. A, Coimmunoprecipitation analysis to determine any physical interaction between ETS2 and DLX3. Immunoprecipitation was performed with antibody to DLX3 and Western analysis with affinity-purified ETS2 antibody. Lane 1 (input), Thirty micrograms of control cell lysate; lane 2 (anti-ETS2), ETS2-DLX3 complexes from control cell lysate; lane 3 (control <t>IgG),</t> immunocomplexes collected after the addition of a nonspecific IgG; lane 4 (anti-ETS2), extracts from cells transfected with both ETS2 and DLX3 expression plasmids (3 μg plasmid DNA/dish). B, Detection of ETS2 and DLX3 on Western blots of lysates from the S1 and S4 lines (which stably express POU5F1), from the C1 and C2 lines (which had been transfected with an empty vector at the same time that the S1 and S2 lines had been generated), and a control JAr cell line (JAr). The loading control was ACTB ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (60 μg) in each lane. C, POU5F1 detection in the S1, S4, C1, and C2 cell lines, with ACTB as a loading control in the lower panel . Molecular weight markers (M r × 10 −3 ) are shown on the left .
    Rabbit Igg1 Control, supplied by SouthernBiotech, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems rabbit igg
    Overlay figures of immunofluorescence staining of MMP-2 (A, B), MMP-9 (C, D), TIMP-2 (E, F) or MT1-MMP (G, H) (red colour) and 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (in blue colour). (A, C, E, G) Undifferentiated mesenchymal stem cells; (B, D, F, H) cells after 28 days of adipogenic differentiation. Arrowheads on H show faintly positive staining of MT1-MMP in forming adipocytes. Negative control staining of day 28 differentiated cells with non-immune goat (I) and rabbit <t>IgG</t> (J).
    Rabbit Igg, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 511 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Sino Biological primary rabbit igg antibody
    Overlay figures of immunofluorescence staining of MMP-2 (A, B), MMP-9 (C, D), TIMP-2 (E, F) or MT1-MMP (G, H) (red colour) and 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (in blue colour). (A, C, E, G) Undifferentiated mesenchymal stem cells; (B, D, F, H) cells after 28 days of adipogenic differentiation. Arrowheads on H show faintly positive staining of MT1-MMP in forming adipocytes. Negative control staining of day 28 differentiated cells with non-immune goat (I) and rabbit <t>IgG</t> (J).
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    Thermo Fisher igg2a
    CD4 + CD25 high Treg expanded by IL-2 complexes display suppressor activity. Purified CD4 + CD25 + Treg and purified responder CD41CD25 − T cells (1×10 5 ) from AChR-primed B6 mice treated with isotype control <t>IgG</t> or IL-2 complexes were prepared
    Igg2a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg antibodies
    Purified Antibodies Directed against Celiac and VP-7 Peptides Bind Endomysial Structures Pooled affinity-purified antibodies against celiac (A and D) and VP-7 (B and E) peptides from ten patients bind endomysium. Purified antibodies against the irrelevant control peptide from five patients (C and F). (A–C) Slides stained with FITC conjugated anti-human <t>IgA</t> antibodies. (D–F) Slides stained with FITC conjugated anti-human <t>IgG</t> antibodies.
    Anti Rabbit Igg Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti rabbit igg secondary antibodies
    PARP1 recruits both XRCC1 and TDP1 to Top1-induced damage sites. ( A ) Endogenous PARP1 co-immunoprecipitates TDP1. HCT116 cells were treated with CPT (10 µM for 2 h) alone or with ABT-888 (5 µM for 2 h). Endogenous PARP1 was immunoprecipitated with anti-PARP1 antibody and immune complexes were blotted with <t>anti-PAR</t> or anti-TDP1 antibodies. Blots were subsequently stripped and probed with anti-PARP1 antibody. Control immunoprecipitation with <t>anti-IgG</t> antibody demonstrates the specificity of the reactions. ( B ) TDP1 co-immunoprecipitates PARP1. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells. Following CPT treatment without or with ABT-888 (as in A), TDP1 was immunoprecipitated with anti-Flag antibody and immune complexes were probed with anti-PAR, anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Protein molecular weight markers (kDa) are indicated at right. ( C ) Knocking down PARP1 abrogates the TDP1-XRCC1 interaction. Flag-tagged hTDP1 was ectopically expressed in isogenic HeLa cells stably transfected with PARP1-shRNA or control (Ctr)-shRNA. Following CPT treatment (as in A), ectopic TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were blotted with anti-XRCC1 antibodies. ( D ) TDP1–PARP1 association is not mediated through DNA. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells treated with CPT (as in A). Cell lysates was pretreated with Benzonase® nuclease before co-immunoprecipitation. The immune complexes were probed with anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Migration of protein molecular weight markers (kDa) is indicated at right. Aliquots (10%) of the input show the level of indicated proteins before immunoprecipitation. ( E ) CPT-induced XRCC1 foci are abrogated by ABT-888. Representative immunofluorescence microscopy image of HCT116 cells treated with CPT (1 µM for 3 h) alone or with ABT-888 (5 µM for 3 h). Focal accumulation of PAR-polymers and XRCC1 are shown in green. ( F ) Defective XRCC1 focus formation in HeLa cells stably transfected with PARP1-shRNA. Representative immunofluorescence microscopy image of control-shRNA (Ctr) or PARP1-shRNA HeLa cells were treated with CPT (1 µM for 3 h) and subsequently fixed and immunostained for XRCC1 (green) or PARP1 (red). Nuclei are outlined as dashed white line circle.
    Anti Rabbit Igg Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher secondary anti rabbit igg antibody
    PARP1 recruits both XRCC1 and TDP1 to Top1-induced damage sites. ( A ) Endogenous PARP1 co-immunoprecipitates TDP1. HCT116 cells were treated with CPT (10 µM for 2 h) alone or with ABT-888 (5 µM for 2 h). Endogenous PARP1 was immunoprecipitated with anti-PARP1 antibody and immune complexes were blotted with <t>anti-PAR</t> or anti-TDP1 antibodies. Blots were subsequently stripped and probed with anti-PARP1 antibody. Control immunoprecipitation with <t>anti-IgG</t> antibody demonstrates the specificity of the reactions. ( B ) TDP1 co-immunoprecipitates PARP1. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells. Following CPT treatment without or with ABT-888 (as in A), TDP1 was immunoprecipitated with anti-Flag antibody and immune complexes were probed with anti-PAR, anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Protein molecular weight markers (kDa) are indicated at right. ( C ) Knocking down PARP1 abrogates the TDP1-XRCC1 interaction. Flag-tagged hTDP1 was ectopically expressed in isogenic HeLa cells stably transfected with PARP1-shRNA or control (Ctr)-shRNA. Following CPT treatment (as in A), ectopic TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were blotted with anti-XRCC1 antibodies. ( D ) TDP1–PARP1 association is not mediated through DNA. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells treated with CPT (as in A). Cell lysates was pretreated with Benzonase® nuclease before co-immunoprecipitation. The immune complexes were probed with anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Migration of protein molecular weight markers (kDa) is indicated at right. Aliquots (10%) of the input show the level of indicated proteins before immunoprecipitation. ( E ) CPT-induced XRCC1 foci are abrogated by ABT-888. Representative immunofluorescence microscopy image of HCT116 cells treated with CPT (1 µM for 3 h) alone or with ABT-888 (5 µM for 3 h). Focal accumulation of PAR-polymers and XRCC1 are shown in green. ( F ) Defective XRCC1 focus formation in HeLa cells stably transfected with PARP1-shRNA. Representative immunofluorescence microscopy image of control-shRNA (Ctr) or PARP1-shRNA HeLa cells were treated with CPT (1 µM for 3 h) and subsequently fixed and immunostained for XRCC1 (green) or PARP1 (red). Nuclei are outlined as dashed white line circle.
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    Nordic-Mubio igg2b
    Induction of anti-nucleolar autoantibodies (ANolA) of <t>IgG1</t> and <t>IgG2a</t> isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with silver nitrate (AgNO 3 ). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with AgNO 3 (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of immunoglobulin (Ig)G1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of silver-treated animals, which produce ANolA.
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    Image Search Results


    Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

    Journal: The Journal of Neuroscience

    Article Title: Circulating Insulin-Like Growth Factor I Mediates Effects of Exercise on the Brain

    doi: 10.1523/JNEUROSCI.20-08-02926.2000

    Figure Lengend Snippet: Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

    Article Snippet: The secondary antibodies that were used were biotinylated goat anti-mouse IgG (1:1000) (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit IgG (1:250–1:1000) (Pierce, Rockford, IL).

    Techniques: Injection, Labeling, Staining, Incubation, Expressing

    STAiR1 – a continuous specifically expressed transcript. (A) INA6 cells were restimulated with IL-6 as described in Figure 3 A and chromatin immunoprecipitated (ChIP-ed) for tri-methylated H3K4 and H3K36, respectively. Enrichment compared to an IgG isotype control was assessed by quantitative real-time PCR using primer sets P1, P3, P5 and P6. The location of respective amplicons is shown in Figure 3 A. Strong enrichment for H3K4me3 is observed only within P1, indicating an active promoter region. H3K36me3 shows strong enrichment throughout the STAiR1 transcript. (B) Expression z -score aggregated over STAiR1 expressed after 1 h (STAiR1 short, chr18:41,591,020-41,720,348) or the entire annotated STAiR1 transcript (STAiR1 long). (C) INA6 cells were restimulated with IL-6 as described and induction of STAiR1 was detected using qRT-PCR with primer sets P1 to P6, as shown in Figure 3 A, and using GAPDH for normalization. This expression time course is consistent with the time-dependent elongation of STAiR1 observed in the tiling array data shown in Figure 3 A. (D) Expression of macroRNAs in different tissues, as detected by reverse transcriptase PCR, using GAPDH as a normalization control. Tissue specificity varies strongly between different macroRNAs. STAiR, STAT3-induced RNA; STAT3, signal transducer and activator of transcription-3.

    Journal: Genome Biology

    Article Title: Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs

    doi: 10.1186/gb-2014-15-3-r48

    Figure Lengend Snippet: STAiR1 – a continuous specifically expressed transcript. (A) INA6 cells were restimulated with IL-6 as described in Figure 3 A and chromatin immunoprecipitated (ChIP-ed) for tri-methylated H3K4 and H3K36, respectively. Enrichment compared to an IgG isotype control was assessed by quantitative real-time PCR using primer sets P1, P3, P5 and P6. The location of respective amplicons is shown in Figure 3 A. Strong enrichment for H3K4me3 is observed only within P1, indicating an active promoter region. H3K36me3 shows strong enrichment throughout the STAiR1 transcript. (B) Expression z -score aggregated over STAiR1 expressed after 1 h (STAiR1 short, chr18:41,591,020-41,720,348) or the entire annotated STAiR1 transcript (STAiR1 long). (C) INA6 cells were restimulated with IL-6 as described and induction of STAiR1 was detected using qRT-PCR with primer sets P1 to P6, as shown in Figure 3 A, and using GAPDH for normalization. This expression time course is consistent with the time-dependent elongation of STAiR1 observed in the tiling array data shown in Figure 3 A. (D) Expression of macroRNAs in different tissues, as detected by reverse transcriptase PCR, using GAPDH as a normalization control. Tissue specificity varies strongly between different macroRNAs. STAiR, STAT3-induced RNA; STAT3, signal transducer and activator of transcription-3.

    Article Snippet: Aliquots containing chromatin from 3×106 cells were used for immunoprecipitation with antibodies against H3K4me3 (ab1012), H3K36me3 (ab9050, both Abcam, Cambridge, UK) and rabbit IgG (# 12-370, Millipore, Schwalbach, Germany), as an isotype control.

    Techniques: Immunoprecipitation, Chromatin Immunoprecipitation, Methylation, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

    Autoantibodies and increased B cells and plasmablasts in CD11c-Flip-KO mice. ( a ) Quantification of serum IgG subclasses by ELISA (μg ml −1 ) in the CD11c-Flip-KO (KO) and control mice. ( b ) ELISA quantification of serum RF, ANA and antiCCP (lower panel). Values are presented as OD 450 × serum dilution for RF and anti-CCP and units for ANA. ( c , d ) Immunoblotting of ankle ( c ) and kidney ( d ) homogenates from Rag −/− mice employing individual serum randomly selected from control and CD11c-Flip-KO mice (≥20 weeks) and a duplicated tissue blot stained with Coomassie blue R250. The arrows identify protein bands common between individual CD11c-Flip-KO but not in control mice. The blots are representative of three independent experiments. ( e , f ) Antibodies identified by autoantigen array. ( e ). Heatmap of IgG antibodies to joint-related antigens, generated as the signal-to-noise ratio (SNR) for each antigen (controls n =7, CD11c-Flip-KO mice ≥20 weeks with arthritis, n =8). ( f ) Three autoantibodies in the array were significantly increased following the Bonferroni correction in the CD11c-Flip-KO mice. ( g , h ) pLNs were examined for B cells ( n =12–15 per group) and plasmablasts ( n =6 per group) from mice ≥20 weeks. B cells were defined as CD19 + B220 + and plasmablasts as CD19 + B220 + CD138 + in the CD64 − CD11b − population. ( i ) Correlation (Pearson's) between the number of pLN plasmablasts and the arthritis clinical score for CD11c-Flip-KO mice just before killing. The values represent the mean±1 s.e. (* P

    Journal: Nature Communications

    Article Title: CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis

    doi: 10.1038/ncomms8086

    Figure Lengend Snippet: Autoantibodies and increased B cells and plasmablasts in CD11c-Flip-KO mice. ( a ) Quantification of serum IgG subclasses by ELISA (μg ml −1 ) in the CD11c-Flip-KO (KO) and control mice. ( b ) ELISA quantification of serum RF, ANA and antiCCP (lower panel). Values are presented as OD 450 × serum dilution for RF and anti-CCP and units for ANA. ( c , d ) Immunoblotting of ankle ( c ) and kidney ( d ) homogenates from Rag −/− mice employing individual serum randomly selected from control and CD11c-Flip-KO mice (≥20 weeks) and a duplicated tissue blot stained with Coomassie blue R250. The arrows identify protein bands common between individual CD11c-Flip-KO but not in control mice. The blots are representative of three independent experiments. ( e , f ) Antibodies identified by autoantigen array. ( e ). Heatmap of IgG antibodies to joint-related antigens, generated as the signal-to-noise ratio (SNR) for each antigen (controls n =7, CD11c-Flip-KO mice ≥20 weeks with arthritis, n =8). ( f ) Three autoantibodies in the array were significantly increased following the Bonferroni correction in the CD11c-Flip-KO mice. ( g , h ) pLNs were examined for B cells ( n =12–15 per group) and plasmablasts ( n =6 per group) from mice ≥20 weeks. B cells were defined as CD19 + B220 + and plasmablasts as CD19 + B220 + CD138 + in the CD64 − CD11b − population. ( i ) Correlation (Pearson's) between the number of pLN plasmablasts and the arthritis clinical score for CD11c-Flip-KO mice just before killing. The values represent the mean±1 s.e. (* P

    Article Snippet: RF in serum diluted 1:25 was detected employing 1 μg ml−1 rabbit IgG (Dako, X0903)-coated ELISA plates .

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Generated

    14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells. a) Cell lysates from M17 cells were immunoprecipitated with a polyclonal rabbit antibody against Bax or rabbit IgG, and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. b) Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).

    Journal: PLoS ONE

    Article Title: 14-3-3theta Protects against Neurotoxicity in a Cellular Parkinson's Disease Model through Inhibition of the Apoptotic Factor Bax

    doi: 10.1371/journal.pone.0021720

    Figure Lengend Snippet: 14-3-3θ immunoprecipitates with Bax in M17 dopaminergic cells. a) Cell lysates from M17 cells were immunoprecipitated with a polyclonal rabbit antibody against Bax or rabbit IgG, and resulting immunoprecipitants were blotted with a monoclonal mouse antibody against 14-3-3θ in top blot. Lysate lane on right is shown at a different exposure time than the immunoprecipitant lanes from the same gel. Blot was reprobed with anti-Bax antibody to verify Bax pulldown (bottom blot). 14-3-3θ shows specific immunoprecipitation with Bax. b) Cell lysates from M17 cells stably transfected with empty vector or 14-3-3θ tagged with the V5 epitope tag were immunoprecipitated with a polyclonal antibody against Bax and then immunoblotted against 14-3-3θ. Both endogenous 14-3-3θ (lower band marked by arrow) and exogenous, tagged 14-3-3θ (higher band marked by arrowhead) were immunoprecipitated with Bax from cells overexpressing 14-3-3θ, and the total amount of 14-3-3θ immunoprecipitated was increased in 14-3-3θ cells compared to empty vector control cells. Lysate lanes on right were run on a separate gel from the immunoprecipitant lanes. Blot was reprobed with anti-Bax antibody to verify pulldown of Bax (bottom blot).

    Article Snippet: Lysates were precleared with Protein G Sepharose (Invitrogen) for one hour, and then incubated overnight at 4C with 2 µg of a rabbit polyclonal antibody against Bax (Cell Signaling, Danvers, MA) or rabbit IgG (Cell Signaling).

    Techniques: Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Plasmid Preparation

    Expression of ETS2, DLX3, and POU5F1 in JAr cell lines. A, Coimmunoprecipitation analysis to determine any physical interaction between ETS2 and DLX3. Immunoprecipitation was performed with antibody to DLX3 and Western analysis with affinity-purified ETS2 antibody. Lane 1 (input), Thirty micrograms of control cell lysate; lane 2 (anti-ETS2), ETS2-DLX3 complexes from control cell lysate; lane 3 (control IgG), immunocomplexes collected after the addition of a nonspecific IgG; lane 4 (anti-ETS2), extracts from cells transfected with both ETS2 and DLX3 expression plasmids (3 μg plasmid DNA/dish). B, Detection of ETS2 and DLX3 on Western blots of lysates from the S1 and S4 lines (which stably express POU5F1), from the C1 and C2 lines (which had been transfected with an empty vector at the same time that the S1 and S2 lines had been generated), and a control JAr cell line (JAr). The loading control was ACTB ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (60 μg) in each lane. C, POU5F1 detection in the S1, S4, C1, and C2 cell lines, with ACTB as a loading control in the lower panel . Molecular weight markers (M r × 10 −3 ) are shown on the left .

    Journal: Molecular Endocrinology

    Article Title: Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells

    doi: 10.1210/me.2011-1146

    Figure Lengend Snippet: Expression of ETS2, DLX3, and POU5F1 in JAr cell lines. A, Coimmunoprecipitation analysis to determine any physical interaction between ETS2 and DLX3. Immunoprecipitation was performed with antibody to DLX3 and Western analysis with affinity-purified ETS2 antibody. Lane 1 (input), Thirty micrograms of control cell lysate; lane 2 (anti-ETS2), ETS2-DLX3 complexes from control cell lysate; lane 3 (control IgG), immunocomplexes collected after the addition of a nonspecific IgG; lane 4 (anti-ETS2), extracts from cells transfected with both ETS2 and DLX3 expression plasmids (3 μg plasmid DNA/dish). B, Detection of ETS2 and DLX3 on Western blots of lysates from the S1 and S4 lines (which stably express POU5F1), from the C1 and C2 lines (which had been transfected with an empty vector at the same time that the S1 and S2 lines had been generated), and a control JAr cell line (JAr). The loading control was ACTB ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (60 μg) in each lane. C, POU5F1 detection in the S1, S4, C1, and C2 cell lines, with ACTB as a loading control in the lower panel . Molecular weight markers (M r × 10 −3 ) are shown on the left .

    Article Snippet: Cell lysate containing 136 μg protein was incubated overnight with either 2 μg of anti-ETS2 antibody (sc-351; Santa Cruz Biotechnology) or normal rabbit IgG (Cell Signaling Technologies).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Affinity Purification, Transfection, Plasmid Preparation, Stable Transfection, Generated, Molecular Weight

    POU5F1, ETS2, and DLX3 expression, interaction, and association with the CGA promoter in hESC before and after exposure to conditions that cause trophoblast differentiation. A, Detection of POU5F1, ETS2, and DLX3 on Western blots of lysates from undifferentiated (hESC/FGF2) (lane 1) and differentiated (hESC/BMP4+PD+A83) H1 hESC (lane 2). The loading control was α-tubulin (TUBA) ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (30 μg) for each lane. B, Coimmunoprecipitation of POU5F1 and ETS2 from hESC control lysates. Immunoprecipitation was performed with antibody to ETS2 and Western analysis with anti-POU5F1. Lane 1 (input), 8 μg of lysate protein from undifferentiated hESC; lane 2 (control IgG), immune complex collected after addition of a nonspecific IgG; lane 3 (anti-ETS2), immune complex containing POU5F1 collected after addition of ETS2. Molecular weight markers (M r × 10 −3 ) are shown on the right side of the panels . C, Control ChIP analysis to demonstrate the association of histone H3 with exon 3 of RPL30 in chromatin prepared from control hESC (hESC/FGF2). Lane 1 shows the lack of reaction product when no template was used in the PCR reaction. Lane 2 is reaction product after the addition of 7.8 μg of chromatin but performing immunoprecipitation with a nonspecific IgG. Lane 3 is the reaction product with same amount of chromatin after immunoprecipitation with antihistone H3. Lane 4 is the amplification product obtained from 5 ng chromatin without immunoprecipitation. D, ChIP analyses demonstrating the lack of association of POU5F1 with the CGA promoter in undifferentiated (hESC/FGF2) H1 hESC. The experiment was identical with that in C except the reaction products were obtained after immunoprecipitating input chromatin with anti-POU5F1. E and F, ChIP analyses demonstrating the association or lack thereof of ETS2 (E) and DLX3 (F) with the CGA promoter in undifferentiated (hESC/FGF2, left panels ) and differentiated (hESC/BMP4+PD+A83, right panels ) H1 hESC. In all experiments (C–F), sheared chromatin preparations were exposed to overnight incubation with either control IgG or ChIP quality antibodies. The primers used for PCR were designed to amplify a region of the CGA proximal promoter (−96 to +48) in E and (−189 to −25) in D and F. G, Chromatin prepared from undifferentiated and differentiated hESC. Chromatin was formaldehyde cross-linked and enzymatically digested. DNA was then purified and analyzed by electrophoresis on a 1% agarose gel. Lane 1, DNA (0.24 μg) from control cells (hESC/FGF2); lane 2, DNA (0.14 μg) from differentiated (BMP4+PD+A83) cells. Gels were stained with ethidium bromide. The majority of the enzymatically fragmented chromatin was 150–500 bp in length.

    Journal: Molecular Endocrinology

    Article Title: Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells

    doi: 10.1210/me.2011-1146

    Figure Lengend Snippet: POU5F1, ETS2, and DLX3 expression, interaction, and association with the CGA promoter in hESC before and after exposure to conditions that cause trophoblast differentiation. A, Detection of POU5F1, ETS2, and DLX3 on Western blots of lysates from undifferentiated (hESC/FGF2) (lane 1) and differentiated (hESC/BMP4+PD+A83) H1 hESC (lane 2). The loading control was α-tubulin (TUBA) ( lower panel ). Analyses were performed on separate blots loaded with the same amounts of protein (30 μg) for each lane. B, Coimmunoprecipitation of POU5F1 and ETS2 from hESC control lysates. Immunoprecipitation was performed with antibody to ETS2 and Western analysis with anti-POU5F1. Lane 1 (input), 8 μg of lysate protein from undifferentiated hESC; lane 2 (control IgG), immune complex collected after addition of a nonspecific IgG; lane 3 (anti-ETS2), immune complex containing POU5F1 collected after addition of ETS2. Molecular weight markers (M r × 10 −3 ) are shown on the right side of the panels . C, Control ChIP analysis to demonstrate the association of histone H3 with exon 3 of RPL30 in chromatin prepared from control hESC (hESC/FGF2). Lane 1 shows the lack of reaction product when no template was used in the PCR reaction. Lane 2 is reaction product after the addition of 7.8 μg of chromatin but performing immunoprecipitation with a nonspecific IgG. Lane 3 is the reaction product with same amount of chromatin after immunoprecipitation with antihistone H3. Lane 4 is the amplification product obtained from 5 ng chromatin without immunoprecipitation. D, ChIP analyses demonstrating the lack of association of POU5F1 with the CGA promoter in undifferentiated (hESC/FGF2) H1 hESC. The experiment was identical with that in C except the reaction products were obtained after immunoprecipitating input chromatin with anti-POU5F1. E and F, ChIP analyses demonstrating the association or lack thereof of ETS2 (E) and DLX3 (F) with the CGA promoter in undifferentiated (hESC/FGF2, left panels ) and differentiated (hESC/BMP4+PD+A83, right panels ) H1 hESC. In all experiments (C–F), sheared chromatin preparations were exposed to overnight incubation with either control IgG or ChIP quality antibodies. The primers used for PCR were designed to amplify a region of the CGA proximal promoter (−96 to +48) in E and (−189 to −25) in D and F. G, Chromatin prepared from undifferentiated and differentiated hESC. Chromatin was formaldehyde cross-linked and enzymatically digested. DNA was then purified and analyzed by electrophoresis on a 1% agarose gel. Lane 1, DNA (0.24 μg) from control cells (hESC/FGF2); lane 2, DNA (0.14 μg) from differentiated (BMP4+PD+A83) cells. Gels were stained with ethidium bromide. The majority of the enzymatically fragmented chromatin was 150–500 bp in length.

    Article Snippet: Cell lysate containing 136 μg protein was incubated overnight with either 2 μg of anti-ETS2 antibody (sc-351; Santa Cruz Biotechnology) or normal rabbit IgG (Cell Signaling Technologies).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Molecular Weight, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Incubation, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining

    ChIP analysis to demonstrate the association of ETS2 with the CGA promoter in presence and absence of POU5F1 expression. A, Sheared chromatin prepared from the S4 line (expressing POU5F1; upper panel ) and the C2, nonexpressing line ( lower panel ) were exposed to overnight incubation with no antibody (lane 1), anti-POU5F1 (lane 2), anti-ETS2 (lane 3), and nonspecific IgG (lane 4). Lane 5 is 10% of total input chromatin. The primers used for PCR in A and B were designed to amplify a region of the CGA proximal promoter (−96 to +48) containing the ETS2 binding site. B, Sheared chromatin prepared from a control JAr cell line after it had been transiently transfected with increasing concentrations (shown on right , 0, 2.5, 10, 50, and 200 ng/ml) of a full-length POU5F1 expression plasmid. Lane 1, No antibody; lane 2, anti-ETS2 antibody; lane 3, nonspecific IgG; lane 4, 10% of total input chromatin.

    Journal: Molecular Endocrinology

    Article Title: Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells

    doi: 10.1210/me.2011-1146

    Figure Lengend Snippet: ChIP analysis to demonstrate the association of ETS2 with the CGA promoter in presence and absence of POU5F1 expression. A, Sheared chromatin prepared from the S4 line (expressing POU5F1; upper panel ) and the C2, nonexpressing line ( lower panel ) were exposed to overnight incubation with no antibody (lane 1), anti-POU5F1 (lane 2), anti-ETS2 (lane 3), and nonspecific IgG (lane 4). Lane 5 is 10% of total input chromatin. The primers used for PCR in A and B were designed to amplify a region of the CGA proximal promoter (−96 to +48) containing the ETS2 binding site. B, Sheared chromatin prepared from a control JAr cell line after it had been transiently transfected with increasing concentrations (shown on right , 0, 2.5, 10, 50, and 200 ng/ml) of a full-length POU5F1 expression plasmid. Lane 1, No antibody; lane 2, anti-ETS2 antibody; lane 3, nonspecific IgG; lane 4, 10% of total input chromatin.

    Article Snippet: Cell lysate containing 136 μg protein was incubated overnight with either 2 μg of anti-ETS2 antibody (sc-351; Santa Cruz Biotechnology) or normal rabbit IgG (Cell Signaling Technologies).

    Techniques: Chromatin Immunoprecipitation, Expressing, Incubation, Polymerase Chain Reaction, Binding Assay, Transfection, Plasmid Preparation

    Overlay figures of immunofluorescence staining of MMP-2 (A, B), MMP-9 (C, D), TIMP-2 (E, F) or MT1-MMP (G, H) (red colour) and 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (in blue colour). (A, C, E, G) Undifferentiated mesenchymal stem cells; (B, D, F, H) cells after 28 days of adipogenic differentiation. Arrowheads on H show faintly positive staining of MT1-MMP in forming adipocytes. Negative control staining of day 28 differentiated cells with non-immune goat (I) and rabbit IgG (J).

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Basement membrane collagen type IV expression by human mesenchymal stem cells during adipogenic differentiation

    doi: 10.1111/j.1582-4934.2011.01442.x

    Figure Lengend Snippet: Overlay figures of immunofluorescence staining of MMP-2 (A, B), MMP-9 (C, D), TIMP-2 (E, F) or MT1-MMP (G, H) (red colour) and 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (in blue colour). (A, C, E, G) Undifferentiated mesenchymal stem cells; (B, D, F, H) cells after 28 days of adipogenic differentiation. Arrowheads on H show faintly positive staining of MT1-MMP in forming adipocytes. Negative control staining of day 28 differentiated cells with non-immune goat (I) and rabbit IgG (J).

    Article Snippet: Corresponding non-immune goat IgG (Jackson ImmunoResearch, West Grove, PA), mouse IgG1/κ (Abcam) or rabbit IgG (R & D Systems) were used at the same concentration as and instead of the primary antibodies as negative staining controls.

    Techniques: Immunofluorescence, Staining, Negative Control

    CD4 + CD25 high Treg expanded by IL-2 complexes display suppressor activity. Purified CD4 + CD25 + Treg and purified responder CD41CD25 − T cells (1×10 5 ) from AChR-primed B6 mice treated with isotype control IgG or IL-2 complexes were prepared

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: CD4 + CD25 high Treg expanded by IL-2 complexes display suppressor activity. Purified CD4 + CD25 + Treg and purified responder CD41CD25 − T cells (1×10 5 ) from AChR-primed B6 mice treated with isotype control IgG or IL-2 complexes were prepared

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Activity Assay, Purification, Mouse Assay

    The profile of IL-2 complex-expanded CD4 + CD25 high Treg in Foxp3 gfp mice. Lymphocytes from AChR-immunized Foxp3 gfp mouse draining lymph nodes treated with isotype control IgG, IL-2 alone, anti-IL-2 mAb alone or IL-2 complexes were prepared on day 35 after

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: The profile of IL-2 complex-expanded CD4 + CD25 high Treg in Foxp3 gfp mice. Lymphocytes from AChR-immunized Foxp3 gfp mouse draining lymph nodes treated with isotype control IgG, IL-2 alone, anti-IL-2 mAb alone or IL-2 complexes were prepared on day 35 after

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Mouse Assay

    IL-2 complexes expand Treg via proliferation of peripheral CD4 + CD25 + T cells and conversion of extrathymic CD4 + CD25 − T cells. On day 35 after immunization, thymocytes from the AChR-immunized Foxp3 gfp mice treated with isotype control IgG or IL-2

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: IL-2 complexes expand Treg via proliferation of peripheral CD4 + CD25 + T cells and conversion of extrathymic CD4 + CD25 − T cells. On day 35 after immunization, thymocytes from the AChR-immunized Foxp3 gfp mice treated with isotype control IgG or IL-2

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Mouse Assay

    IL-2 complexes expand Treg in thymectomized Foxp3 gfp mice. Foxp3 gfp mice were thymectomized and then immunized with AChR/CFA, treated with isotype control IgG or IL-2 complexes. On day 35 after immunization, lymphocytes were prepared from these Foxp3

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: IL-2 complexes expand Treg in thymectomized Foxp3 gfp mice. Foxp3 gfp mice were thymectomized and then immunized with AChR/CFA, treated with isotype control IgG or IL-2 complexes. On day 35 after immunization, lymphocytes were prepared from these Foxp3

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Mouse Assay

    Impact of IL-2 complexes on EAMG. Groups of B6 mice treated with isotype control IgG or IL-2 complexes were immunized with AChR and monitored for clinical scores of muscle weakness as described in the Materials and methods section. (A) Treatment started

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: Impact of IL-2 complexes on EAMG. Groups of B6 mice treated with isotype control IgG or IL-2 complexes were immunized with AChR and monitored for clinical scores of muscle weakness as described in the Materials and methods section. (A) Treatment started

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Mouse Assay

    Homeostasis of CD4 + CD25 high Treg in AChR-primed mice treated with IL-2 complexes. Splenocytes from AChR-immunized B6 mice treated with isotype control IgG or IL-2 complexes were prepared on the indicated days after immunization, and stained with anti-CD4

    Journal: European journal of immunology

    Article Title: Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

    doi: 10.1002/eji.200939792

    Figure Lengend Snippet: Homeostasis of CD4 + CD25 high Treg in AChR-primed mice treated with IL-2 complexes. Splenocytes from AChR-immunized B6 mice treated with isotype control IgG or IL-2 complexes were prepared on the indicated days after immunization, and stained with anti-CD4

    Article Snippet: HRP-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM (1/1000, Invitrogen, Carlsbad, CA, USA) were added and followed by color development with TMB (3, 3′, 5, 5′-tetramethylbenzidine) substrate.

    Techniques: Mouse Assay, Staining

    Purified Antibodies Directed against Celiac and VP-7 Peptides Bind Endomysial Structures Pooled affinity-purified antibodies against celiac (A and D) and VP-7 (B and E) peptides from ten patients bind endomysium. Purified antibodies against the irrelevant control peptide from five patients (C and F). (A–C) Slides stained with FITC conjugated anti-human IgA antibodies. (D–F) Slides stained with FITC conjugated anti-human IgG antibodies.

    Journal: PLoS Medicine

    Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

    doi: 10.1371/journal.pmed.0030358

    Figure Lengend Snippet: Purified Antibodies Directed against Celiac and VP-7 Peptides Bind Endomysial Structures Pooled affinity-purified antibodies against celiac (A and D) and VP-7 (B and E) peptides from ten patients bind endomysium. Purified antibodies against the irrelevant control peptide from five patients (C and F). (A–C) Slides stained with FITC conjugated anti-human IgA antibodies. (D–F) Slides stained with FITC conjugated anti-human IgG antibodies.

    Article Snippet: Blots were probed with primary antibodies followed by either peroxidase-linked anti-human Igs antibodies, anti-human IgA antibodies, anti-rabbit IgG antibodies, or anti-mouse IgG antibodies (all purchased from Sigma).

    Techniques: Purification, Affinity Purification, Staining

    Celiac Peptide Is Recognized by Sera of Patients with Active Disease and Shares Homology with Microbial Antigens (A) The celiac peptide is recognized by serum IgA immunoglobulins of patients on GCD, but not by patients on GFD. Results are expressed as absorbance at 405 nm. (B) Sequence homology between the celiac peptide and infectious agents. The peptide sequence was compared with known microbial protein sequences using the BLASTP via the NCBI BLAST network service (: indicates identity and * indicates conservative substitutions). (C) Frequency of IgG antibodies directed against infectious agents in patients with active CD and in normal healthy controls. (D) Sera of patients with active CD contain IgA antibodies directed against the rotavirus major neutralizing protein VP-7. A rotavirus extract was probed with rabbit antiserum raised against a peptide (VIQVGGSNVLDI) of the VP-7 protein (Lane 1), with affinity-purified anti-celiac peptide antibodies (Lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from adult patients with active disease (Lanes 4 and 5), with sera from the same patients on GFD (Lanes 7 and 8), and serum from a 1-y-old child with active CD (Lane 6) and on GFD (Lane 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection.

    Journal: PLoS Medicine

    Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

    doi: 10.1371/journal.pmed.0030358

    Figure Lengend Snippet: Celiac Peptide Is Recognized by Sera of Patients with Active Disease and Shares Homology with Microbial Antigens (A) The celiac peptide is recognized by serum IgA immunoglobulins of patients on GCD, but not by patients on GFD. Results are expressed as absorbance at 405 nm. (B) Sequence homology between the celiac peptide and infectious agents. The peptide sequence was compared with known microbial protein sequences using the BLASTP via the NCBI BLAST network service (: indicates identity and * indicates conservative substitutions). (C) Frequency of IgG antibodies directed against infectious agents in patients with active CD and in normal healthy controls. (D) Sera of patients with active CD contain IgA antibodies directed against the rotavirus major neutralizing protein VP-7. A rotavirus extract was probed with rabbit antiserum raised against a peptide (VIQVGGSNVLDI) of the VP-7 protein (Lane 1), with affinity-purified anti-celiac peptide antibodies (Lane 2), with antibodies affinity-purified against an irrelevant control peptide (Lane 3), with sera from adult patients with active disease (Lanes 4 and 5), with sera from the same patients on GFD (Lanes 7 and 8), and serum from a 1-y-old child with active CD (Lane 6) and on GFD (Lane 9). A peroxidase-labelled polyvalent anti-human Igs antibody (Lanes 2 and 3) and an anti-human IgA antibody (Lanes 4–9) were used for detection.

    Article Snippet: Blots were probed with primary antibodies followed by either peroxidase-linked anti-human Igs antibodies, anti-human IgA antibodies, anti-rabbit IgG antibodies, or anti-mouse IgG antibodies (all purchased from Sigma).

    Techniques: Sequencing, Affinity Purification

    Pro-Inflammatory Cytokines Produced by Activated Monocytes Levels of IL-6 (A), IL-12 (B), and TNF-alpha (C) released in the supernatant by monocytes incubated with medium alone (Line 1), with antibodies directed against an irrelevant peptide (Line 2), with LPS (Line 3), with pooled Igs isolated from the 22 patients with active CD (Line 4), with pooled Igs isolated from the same patients on GFD (Line 5), with purified anti-celiac peptide antibodies obtained from ten patients (line 6), with purified anti-celiac peptide antibodies in the presence of an irrelevant mouse IgG2b antibody (20 μg/ml) (Line 7), and with purified anti-celiac peptide antibodies in the presence of the neutralizing mouse monoclonal antibody anti-TLR4, clone HTA 125 (20 μg/ml) (Line 8). The y -axis represents the cytokine concentration expressed as pg/ml. Data represent the mean ±SD of three independently performed experiments.

    Journal: PLoS Medicine

    Article Title: In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

    doi: 10.1371/journal.pmed.0030358

    Figure Lengend Snippet: Pro-Inflammatory Cytokines Produced by Activated Monocytes Levels of IL-6 (A), IL-12 (B), and TNF-alpha (C) released in the supernatant by monocytes incubated with medium alone (Line 1), with antibodies directed against an irrelevant peptide (Line 2), with LPS (Line 3), with pooled Igs isolated from the 22 patients with active CD (Line 4), with pooled Igs isolated from the same patients on GFD (Line 5), with purified anti-celiac peptide antibodies obtained from ten patients (line 6), with purified anti-celiac peptide antibodies in the presence of an irrelevant mouse IgG2b antibody (20 μg/ml) (Line 7), and with purified anti-celiac peptide antibodies in the presence of the neutralizing mouse monoclonal antibody anti-TLR4, clone HTA 125 (20 μg/ml) (Line 8). The y -axis represents the cytokine concentration expressed as pg/ml. Data represent the mean ±SD of three independently performed experiments.

    Article Snippet: Blots were probed with primary antibodies followed by either peroxidase-linked anti-human Igs antibodies, anti-human IgA antibodies, anti-rabbit IgG antibodies, or anti-mouse IgG antibodies (all purchased from Sigma).

    Techniques: Produced, Incubation, Isolation, Purification, Concentration Assay

    PARP1 recruits both XRCC1 and TDP1 to Top1-induced damage sites. ( A ) Endogenous PARP1 co-immunoprecipitates TDP1. HCT116 cells were treated with CPT (10 µM for 2 h) alone or with ABT-888 (5 µM for 2 h). Endogenous PARP1 was immunoprecipitated with anti-PARP1 antibody and immune complexes were blotted with anti-PAR or anti-TDP1 antibodies. Blots were subsequently stripped and probed with anti-PARP1 antibody. Control immunoprecipitation with anti-IgG antibody demonstrates the specificity of the reactions. ( B ) TDP1 co-immunoprecipitates PARP1. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells. Following CPT treatment without or with ABT-888 (as in A), TDP1 was immunoprecipitated with anti-Flag antibody and immune complexes were probed with anti-PAR, anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Protein molecular weight markers (kDa) are indicated at right. ( C ) Knocking down PARP1 abrogates the TDP1-XRCC1 interaction. Flag-tagged hTDP1 was ectopically expressed in isogenic HeLa cells stably transfected with PARP1-shRNA or control (Ctr)-shRNA. Following CPT treatment (as in A), ectopic TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were blotted with anti-XRCC1 antibodies. ( D ) TDP1–PARP1 association is not mediated through DNA. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells treated with CPT (as in A). Cell lysates was pretreated with Benzonase® nuclease before co-immunoprecipitation. The immune complexes were probed with anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Migration of protein molecular weight markers (kDa) is indicated at right. Aliquots (10%) of the input show the level of indicated proteins before immunoprecipitation. ( E ) CPT-induced XRCC1 foci are abrogated by ABT-888. Representative immunofluorescence microscopy image of HCT116 cells treated with CPT (1 µM for 3 h) alone or with ABT-888 (5 µM for 3 h). Focal accumulation of PAR-polymers and XRCC1 are shown in green. ( F ) Defective XRCC1 focus formation in HeLa cells stably transfected with PARP1-shRNA. Representative immunofluorescence microscopy image of control-shRNA (Ctr) or PARP1-shRNA HeLa cells were treated with CPT (1 µM for 3 h) and subsequently fixed and immunostained for XRCC1 (green) or PARP1 (red). Nuclei are outlined as dashed white line circle.

    Journal: Nucleic Acids Research

    Article Title: PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage

    doi: 10.1093/nar/gku088

    Figure Lengend Snippet: PARP1 recruits both XRCC1 and TDP1 to Top1-induced damage sites. ( A ) Endogenous PARP1 co-immunoprecipitates TDP1. HCT116 cells were treated with CPT (10 µM for 2 h) alone or with ABT-888 (5 µM for 2 h). Endogenous PARP1 was immunoprecipitated with anti-PARP1 antibody and immune complexes were blotted with anti-PAR or anti-TDP1 antibodies. Blots were subsequently stripped and probed with anti-PARP1 antibody. Control immunoprecipitation with anti-IgG antibody demonstrates the specificity of the reactions. ( B ) TDP1 co-immunoprecipitates PARP1. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells. Following CPT treatment without or with ABT-888 (as in A), TDP1 was immunoprecipitated with anti-Flag antibody and immune complexes were probed with anti-PAR, anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Protein molecular weight markers (kDa) are indicated at right. ( C ) Knocking down PARP1 abrogates the TDP1-XRCC1 interaction. Flag-tagged hTDP1 was ectopically expressed in isogenic HeLa cells stably transfected with PARP1-shRNA or control (Ctr)-shRNA. Following CPT treatment (as in A), ectopic TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were blotted with anti-XRCC1 antibodies. ( D ) TDP1–PARP1 association is not mediated through DNA. Flag-tagged hTDP1 was ectopically expressed in HCT116 cells treated with CPT (as in A). Cell lysates was pretreated with Benzonase® nuclease before co-immunoprecipitation. The immune complexes were probed with anti-PARP1 or anti-XRCC1 antibodies. Blots were subsequently stripped and probed with anti-TDP1 antibody. Migration of protein molecular weight markers (kDa) is indicated at right. Aliquots (10%) of the input show the level of indicated proteins before immunoprecipitation. ( E ) CPT-induced XRCC1 foci are abrogated by ABT-888. Representative immunofluorescence microscopy image of HCT116 cells treated with CPT (1 µM for 3 h) alone or with ABT-888 (5 µM for 3 h). Focal accumulation of PAR-polymers and XRCC1 are shown in green. ( F ) Defective XRCC1 focus formation in HeLa cells stably transfected with PARP1-shRNA. Representative immunofluorescence microscopy image of control-shRNA (Ctr) or PARP1-shRNA HeLa cells were treated with CPT (1 µM for 3 h) and subsequently fixed and immunostained for XRCC1 (green) or PARP1 (red). Nuclei are outlined as dashed white line circle.

    Article Snippet: Following 10-min fixation with 4% paraformaldehyde at room temperature, primary antibodies against PAR, PARP1 and XRCC1 were detected with anti-mouse or anti-rabbit IgG secondary antibodies labeled with Alexa 488/568 (Invitrogen).

    Techniques: Cycling Probe Technology, Immunoprecipitation, Molecular Weight, Stable Transfection, Transfection, shRNA, Migration, Immunofluorescence, Microscopy

    Direct interaction between the N-terminus of TDP1 and the C-terminus of PARP1. ( A ) His 6 -tagged hTDP1 pulls down recombinant human PARP1 (hPARP1). L1, L2 and L3: Loaded samples following 1-h incubation at 4°C (reaction conditions are indicated at the bottom). W1, W2 and W3: excess unbound proteins after washing the Ni 2+ -NTA-agarose beads with 50 mM imidazole. E1, E2 and E3: bound proteins eluted with 300 mM imidazole. Right panel: control reactions showing that hPARP1 alone (L4) or in the presence of DNA plus NAD + (L5) does not bind the Ni 2+ -NTA-agarose beads and is recovered in the flow through (Ft). W4, W5 and E4, E5 are washed and eluted fractions with 50 and 300 mM imidazole, respectively. PARP1 and TDP1 were detected by Western blotting after 8% SDS-PAGE. ( B ) Schematic representation of Flag-tagged full-length hTDP1 and truncated hTDP1 (residues 1–185) constructs. The regulatory NTD and the catalytic domain are indicated with different colors. ( C ) PARP1 binds the NTD of TDP1. Flag-tagged TDP1 constructs were expressed in HCT116 cells. TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were probed with anti-PARP1 antibody. Blots were subsequently stripped and probed with anti-Flag antibody. Lower panel shows PARP1 input corresponding to one-tenth of the lysate. Control immunoprecipitation with anti-IgG demonstrates the specificity of the reactions. ( D ) Schematic representation of GST-fused full-length hPARP1 and hPARP1 truncated domains corresponding to the DNA binding domain (DBD) (residues 1–371), the BRCT (C-terminal domain of a breast cancer susceptibility protein; residues 384–524) and the C-terminal domain (CTD) harboring the catalytic site (residues 525–1014). ( E ) TDP1 binds the CTD of PARP1. The GST-tagged full-length and truncated domains of PARP1 were expressed in HCT116 cells. PARP1 and its truncated domains were immunoprecipitated using anti-GST antibody, and the immune complexes were detected by western blotting after 4–20% SDS-PAGE with anti-TDP1 antibody. Lower panel shows TDP1 input corresponding to one-tenth of the lysates.

    Journal: Nucleic Acids Research

    Article Title: PARP1-TDP1 coupling for the repair of topoisomerase I-induced DNA damage

    doi: 10.1093/nar/gku088

    Figure Lengend Snippet: Direct interaction between the N-terminus of TDP1 and the C-terminus of PARP1. ( A ) His 6 -tagged hTDP1 pulls down recombinant human PARP1 (hPARP1). L1, L2 and L3: Loaded samples following 1-h incubation at 4°C (reaction conditions are indicated at the bottom). W1, W2 and W3: excess unbound proteins after washing the Ni 2+ -NTA-agarose beads with 50 mM imidazole. E1, E2 and E3: bound proteins eluted with 300 mM imidazole. Right panel: control reactions showing that hPARP1 alone (L4) or in the presence of DNA plus NAD + (L5) does not bind the Ni 2+ -NTA-agarose beads and is recovered in the flow through (Ft). W4, W5 and E4, E5 are washed and eluted fractions with 50 and 300 mM imidazole, respectively. PARP1 and TDP1 were detected by Western blotting after 8% SDS-PAGE. ( B ) Schematic representation of Flag-tagged full-length hTDP1 and truncated hTDP1 (residues 1–185) constructs. The regulatory NTD and the catalytic domain are indicated with different colors. ( C ) PARP1 binds the NTD of TDP1. Flag-tagged TDP1 constructs were expressed in HCT116 cells. TDP1 was immunoprecipitated using anti-Flag antibody and the immune complexes were probed with anti-PARP1 antibody. Blots were subsequently stripped and probed with anti-Flag antibody. Lower panel shows PARP1 input corresponding to one-tenth of the lysate. Control immunoprecipitation with anti-IgG demonstrates the specificity of the reactions. ( D ) Schematic representation of GST-fused full-length hPARP1 and hPARP1 truncated domains corresponding to the DNA binding domain (DBD) (residues 1–371), the BRCT (C-terminal domain of a breast cancer susceptibility protein; residues 384–524) and the C-terminal domain (CTD) harboring the catalytic site (residues 525–1014). ( E ) TDP1 binds the CTD of PARP1. The GST-tagged full-length and truncated domains of PARP1 were expressed in HCT116 cells. PARP1 and its truncated domains were immunoprecipitated using anti-GST antibody, and the immune complexes were detected by western blotting after 4–20% SDS-PAGE with anti-TDP1 antibody. Lower panel shows TDP1 input corresponding to one-tenth of the lysates.

    Article Snippet: Following 10-min fixation with 4% paraformaldehyde at room temperature, primary antibodies against PAR, PARP1 and XRCC1 were detected with anti-mouse or anti-rabbit IgG secondary antibodies labeled with Alexa 488/568 (Invitrogen).

    Techniques: Recombinant, Incubation, Flow Cytometry, Western Blot, SDS Page, Construct, Immunoprecipitation, Binding Assay

    Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with silver nitrate (AgNO 3 ). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with AgNO 3 (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of immunoglobulin (Ig)G1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of silver-treated animals, which produce ANolA.

    Journal: Clinical and Experimental Immunology

    Article Title: Mercury and silver induce B cell activation and anti-nucleolar autoantibody production in outbred mouse stocks: are environmental factors more important than the susceptibility genes in connection with autoimmunity?

    doi: 10.1111/j.1365-2249.2008.03801.x

    Figure Lengend Snippet: Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with silver nitrate (AgNO 3 ). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with AgNO 3 (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of immunoglobulin (Ig)G1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of silver-treated animals, which produce ANolA.

    Article Snippet: The numbers of splenic cells secreting antibodies belonging to different Ig classes and subclasses were quantified utilizing the protein A plaque assay described by Gronowicz et al. [ ], employing rabbit anti-mouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC, USA) and IgG2b (Nordic Immunological Laboratories, Tillburg, the Netherlands) as the developing reagents.

    Techniques: Mouse Assay, Injection, Immunofluorescence

    Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with mercuric chloride (HgCl 2 ). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of IgG1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of mercury-treated animals, which produce ANolA.

    Journal: Clinical and Experimental Immunology

    Article Title: Mercury and silver induce B cell activation and anti-nucleolar autoantibody production in outbred mouse stocks: are environmental factors more important than the susceptibility genes in connection with autoimmunity?

    doi: 10.1111/j.1365-2249.2008.03801.x

    Figure Lengend Snippet: Induction of anti-nucleolar autoantibodies (ANolA) of IgG1 and IgG2a isotypes in outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mice treated with mercuric chloride (HgCl 2 ). Groups of female ICR (solid and open circles), NMRI (solid and open squares) and Black Swiss (solid and open hexagons) outbred mice were injected repeatedly subcutaneously (s.c.) with HgCl 2 (solid symbols) or NaCl (open symbols) for 4 weeks. At the end of each experiment the mice were bled and killed. The sera were tested for the presence of IgG1 (a) and IgG2a (b) ANolA by using an indirect immunofluorescence (IIF) method. Each symbol represents a single animal. The mean values ± standard error are presented as thin vertical lines. Values in parentheses represent the proportions of mercury-treated animals, which produce ANolA.

    Article Snippet: The numbers of splenic cells secreting antibodies belonging to different Ig classes and subclasses were quantified utilizing the protein A plaque assay described by Gronowicz et al. [ ], employing rabbit anti-mouse IgM, IgG1, IgG3 (Organon Teknika, Durham, NC, USA) and IgG2b (Nordic Immunological Laboratories, Tillburg, the Netherlands) as the developing reagents.

    Techniques: Mouse Assay, Injection, Immunofluorescence