immunoprecipitation kit  (Roche)


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    Structured Review

    Roche immunoprecipitation kit
    Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by <t>co-immunoprecipitation</t> using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.
    Immunoprecipitation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis"

    Article Title: Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis

    Journal: Cell death and differentiation

    doi: 10.1038/cdd.2009.86

    Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by co-immunoprecipitation using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.
    Figure Legend Snippet: Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by co-immunoprecipitation using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.

    Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Transfection

    2) Product Images from "Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex"

    Article Title: Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052640

    Schematic overview of the SILAC approach. “Heavy”-labeled cells were co-transfected with the plasmids CHD7-CR1-3-pCMV-HA (containing amino acids 1593-2178, NP_060250.2, in fusion with an HA-tag) and CHD8-pCMV-cmyc (spanning amino acids 1789–2302, NP_065971.2, in fusion with a cmyc-tag). The CHD7 part was purified by anti-HA immunoprecipitation. As a negative control, the same immunoprecipitation was performed in lysates of non-transfected “Light”-labeled HeLa cells. Purified proteins from both cell cultures were pooled in equimolar amounts and in-gel digested, followed by liquid-chromatography (LC)-coupled tandem mass spectrometry.
    Figure Legend Snippet: Schematic overview of the SILAC approach. “Heavy”-labeled cells were co-transfected with the plasmids CHD7-CR1-3-pCMV-HA (containing amino acids 1593-2178, NP_060250.2, in fusion with an HA-tag) and CHD8-pCMV-cmyc (spanning amino acids 1789–2302, NP_065971.2, in fusion with a cmyc-tag). The CHD7 part was purified by anti-HA immunoprecipitation. As a negative control, the same immunoprecipitation was performed in lysates of non-transfected “Light”-labeled HeLa cells. Purified proteins from both cell cultures were pooled in equimolar amounts and in-gel digested, followed by liquid-chromatography (LC)-coupled tandem mass spectrometry.

    Techniques Used: Labeling, Transfection, Purification, Immunoprecipitation, Negative Control, Liquid Chromatography, Mass Spectrometry

    Co-immunoprecipitation of FAM124B with a part of CHD7 and CHD8. HeLa cells were co-transfected with either the CHD7-CR1-3-pCMV-HA (amino acids 1593-2178, NP_060250.2) plasmid and FAM124B-1,3-pCMV-cmyc or FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1) or with CHD8-pCMV-cmyc (amino acids 1789-2302, NP_065971.2) and FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1). ( A ) Using the anti-CHD8 (abcam, ab84527) or the anti-CHD7 (abcam, ab31824) antibody for precipitation, we detected with the anti-HA antibody (Roche) an approximately 51 kDa band corresponding to the estimated size of FAM124B transcript variant 1. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( B ) Reciprocal immunoprecipitation with anti-cmyc antibody (precipitating FAM124B transcript variant 1), and detection with the anti-CHD7 antibody lead to a specific band ∼70 kDa, the estimated size for the CHD7 part fused to the HA-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( C ) Reciprocal experiment with anti-HA antibody (precipitating FAM124B transcript variant 1) and detection with the anti-CHD8 antibody detected a specific band ∼68 kDa, the estimated size for the CHD8 part fused to the cmyc-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( D, E, F ) The same experimental procedure was performed for FAM124B transcript variant 2, demonstrating a specific interaction of FAM124B transcript variant 2 with the CHD7 and CHD8 part as well. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control.
    Figure Legend Snippet: Co-immunoprecipitation of FAM124B with a part of CHD7 and CHD8. HeLa cells were co-transfected with either the CHD7-CR1-3-pCMV-HA (amino acids 1593-2178, NP_060250.2) plasmid and FAM124B-1,3-pCMV-cmyc or FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1) or with CHD8-pCMV-cmyc (amino acids 1789-2302, NP_065971.2) and FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1). ( A ) Using the anti-CHD8 (abcam, ab84527) or the anti-CHD7 (abcam, ab31824) antibody for precipitation, we detected with the anti-HA antibody (Roche) an approximately 51 kDa band corresponding to the estimated size of FAM124B transcript variant 1. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( B ) Reciprocal immunoprecipitation with anti-cmyc antibody (precipitating FAM124B transcript variant 1), and detection with the anti-CHD7 antibody lead to a specific band ∼70 kDa, the estimated size for the CHD7 part fused to the HA-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( C ) Reciprocal experiment with anti-HA antibody (precipitating FAM124B transcript variant 1) and detection with the anti-CHD8 antibody detected a specific band ∼68 kDa, the estimated size for the CHD8 part fused to the cmyc-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( D, E, F ) The same experimental procedure was performed for FAM124B transcript variant 2, demonstrating a specific interaction of FAM124B transcript variant 2 with the CHD7 and CHD8 part as well. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control.

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Variant Assay, Co-Immunoprecipitation Assay, Negative Control

    3) Product Images from "Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications"

    Article Title: Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-14-19

    SDS-PAGE analysis of secretory expression of NFG1CG4-hFasLECD. a) Comparison of the expression level using glass baffled-flask with that using plastic culture-bag. Lanes: M, molecular-weight size-markers; a and b, at 0 h; c and d, at 24 h; e and f, at 48 h; g and h, at 72 h; i and j, at 96 h. Five μl each samples of culture supernatants either from 500 ml scale culture in 3000 ml baffled flask (lanes a, c, e, g and i) or 2500 ml scale culture in 5000 ml disposable plastic bag (lanes b, d, f, h and j) were applied to each lane. Upper and lower arrows indicate the migration positions of dimeric and monomeric subunits of NFG1CG4-hFasLECD, respectively. b) SDS-PAGE analysis of disulfide bond reduction in partially purified NFG1CG4-hFasLECD. Main peak fraction of the P. pastoris culture supernatant (29.5°C, 96 h) in 1st cation-exchange column chromatography was concentrated and treated with SDS-PAGE sample buffers with / without 2-mercaptoethanol. Lanes: M, molecular-weight size markers; a, with 2-mercaptoethanol; b, without 2-mercaptoethanol. Upper and lower arrows indicate the same as in a) . c) Co-immunoprecipitation of the secreted product with wild-type hFasRECD-T-Fc. Lanes: M, molecular-weight size-markers; a, 5 μl culture supernatant of 40-fold concentrated NFG1CG4-hFasLECD from 500 ml scale culture in 3000 ml baffled flask at 96 h; b, purified NFG5-hFasLECD [ 10 ] (5 μg); c, buffer alone. Upper and lower arrows indicate the same as in a) . The bands in “FasR-Fc” labeled region were derived from wild-type hFasRECD-T-Fc sample.
    Figure Legend Snippet: SDS-PAGE analysis of secretory expression of NFG1CG4-hFasLECD. a) Comparison of the expression level using glass baffled-flask with that using plastic culture-bag. Lanes: M, molecular-weight size-markers; a and b, at 0 h; c and d, at 24 h; e and f, at 48 h; g and h, at 72 h; i and j, at 96 h. Five μl each samples of culture supernatants either from 500 ml scale culture in 3000 ml baffled flask (lanes a, c, e, g and i) or 2500 ml scale culture in 5000 ml disposable plastic bag (lanes b, d, f, h and j) were applied to each lane. Upper and lower arrows indicate the migration positions of dimeric and monomeric subunits of NFG1CG4-hFasLECD, respectively. b) SDS-PAGE analysis of disulfide bond reduction in partially purified NFG1CG4-hFasLECD. Main peak fraction of the P. pastoris culture supernatant (29.5°C, 96 h) in 1st cation-exchange column chromatography was concentrated and treated with SDS-PAGE sample buffers with / without 2-mercaptoethanol. Lanes: M, molecular-weight size markers; a, with 2-mercaptoethanol; b, without 2-mercaptoethanol. Upper and lower arrows indicate the same as in a) . c) Co-immunoprecipitation of the secreted product with wild-type hFasRECD-T-Fc. Lanes: M, molecular-weight size-markers; a, 5 μl culture supernatant of 40-fold concentrated NFG1CG4-hFasLECD from 500 ml scale culture in 3000 ml baffled flask at 96 h; b, purified NFG5-hFasLECD [ 10 ] (5 μg); c, buffer alone. Upper and lower arrows indicate the same as in a) . The bands in “FasR-Fc” labeled region were derived from wild-type hFasRECD-T-Fc sample.

    Techniques Used: SDS Page, Expressing, Molecular Weight, Migration, Purification, Column Chromatography, Immunoprecipitation, Labeling, Derivative Assay

    SDS-PAGE analysis of hFasRECD-T-Fc mediated co-immunoprecipitation of the fractionated hFasLECDs cross-linked with SUNBRIGHT ® DE-100MA. Either wild-type hFasRECD-T-Fc (lanes a and c) or hFasRECD-T-Fc (N102Q, N120Q) mutant (lanes b and d) was used for the experiment. Lanes: M, molecular-weight size markers; a and b, peak II (co-immunoprecipitated sample); c and d, peak III (co-immunoprecipitated sample); e, peak II (fractionated sample, 0.3 μg); f, peak III (fractionated sample, 0.3 μg). The components in the peaks II and III are arrowed.
    Figure Legend Snippet: SDS-PAGE analysis of hFasRECD-T-Fc mediated co-immunoprecipitation of the fractionated hFasLECDs cross-linked with SUNBRIGHT ® DE-100MA. Either wild-type hFasRECD-T-Fc (lanes a and c) or hFasRECD-T-Fc (N102Q, N120Q) mutant (lanes b and d) was used for the experiment. Lanes: M, molecular-weight size markers; a and b, peak II (co-immunoprecipitated sample); c and d, peak III (co-immunoprecipitated sample); e, peak II (fractionated sample, 0.3 μg); f, peak III (fractionated sample, 0.3 μg). The components in the peaks II and III are arrowed.

    Techniques Used: SDS Page, Immunoprecipitation, Mutagenesis, Molecular Weight

    Purification of N-glycan untrimmed and trimmed tag-free hFasLECDs. a) Elution profile of N-glycan untrimmed tag-free hFasLECD sample (pre-purified by 1st Hi-Trap S cation-exchange chromatography) in 2nd cation-exchange chromatography. Used column: Resource S 6 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. b) Elution profile of N-glycan trimmed tag-free hFasLECD sample in 3rd cation-exchange chromatography. Used column: Mono S 1 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. c) Elution profiles of fractionated products in size-exclusion chromatography. Solid line: N-glycan untrimmed tag-free hFasLECD. Dashed line: N-glycan trimmed tag-free hFasLECD. Used column: Superdex 200 10/300 GL. Elution buffer: 50 mM sodium acetate plus 150 mM NaCl (pH 5.6). Flow rate: 0.5 ml/min. The peak retention time of each sample is described. Vertical arrows indicate the elution positions of molecular-weight size-markers [Ald, aldolase (158 kDa); Ova, ovalbumin (44 kDa); Rna, ribonuclease A (13.7 kDa) under the same conditions. d) SDS-PAGE analysis of purification course during N-glycan trimming with Endo Hf glycosidase and receptor-mediated co-immunoprecipitation using wild type and mutant hFasRECD-T-Fcs. Lanes: a, N-glycan untrimmed tag-free hFasLECD; b, after Endo Hf digestion; c, after Con A column fractionation; d, after Mono S column fractionation; e, co-immunoprecipitated materials using wild-type hFasRECD-T-Fc [ 15 ]; f, co-immunoprecipitated materials using hFasRECD-T-Fc (N102Q, N120Q) mutant; M, Molecular-weight markers. “+CHO” and “ΔCHO” indicate the migration positions of N-glycan untrimmed and N-glycan trimmed tag-free hFasLECD, respectively.
    Figure Legend Snippet: Purification of N-glycan untrimmed and trimmed tag-free hFasLECDs. a) Elution profile of N-glycan untrimmed tag-free hFasLECD sample (pre-purified by 1st Hi-Trap S cation-exchange chromatography) in 2nd cation-exchange chromatography. Used column: Resource S 6 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. b) Elution profile of N-glycan trimmed tag-free hFasLECD sample in 3rd cation-exchange chromatography. Used column: Mono S 1 ml. The region shown in underbar was collected and used for characterization in c) . NaCl concentration under principal peak eluting condition is described. c) Elution profiles of fractionated products in size-exclusion chromatography. Solid line: N-glycan untrimmed tag-free hFasLECD. Dashed line: N-glycan trimmed tag-free hFasLECD. Used column: Superdex 200 10/300 GL. Elution buffer: 50 mM sodium acetate plus 150 mM NaCl (pH 5.6). Flow rate: 0.5 ml/min. The peak retention time of each sample is described. Vertical arrows indicate the elution positions of molecular-weight size-markers [Ald, aldolase (158 kDa); Ova, ovalbumin (44 kDa); Rna, ribonuclease A (13.7 kDa) under the same conditions. d) SDS-PAGE analysis of purification course during N-glycan trimming with Endo Hf glycosidase and receptor-mediated co-immunoprecipitation using wild type and mutant hFasRECD-T-Fcs. Lanes: a, N-glycan untrimmed tag-free hFasLECD; b, after Endo Hf digestion; c, after Con A column fractionation; d, after Mono S column fractionation; e, co-immunoprecipitated materials using wild-type hFasRECD-T-Fc [ 15 ]; f, co-immunoprecipitated materials using hFasRECD-T-Fc (N102Q, N120Q) mutant; M, Molecular-weight markers. “+CHO” and “ΔCHO” indicate the migration positions of N-glycan untrimmed and N-glycan trimmed tag-free hFasLECD, respectively.

    Techniques Used: Purification, Chromatography, Concentration Assay, Size-exclusion Chromatography, Flow Cytometry, Molecular Weight, SDS Page, Immunoprecipitation, Mutagenesis, Fractionation, Migration

    SDS-PAGE analysis of hFasRECD-T-Fc mediated co-immunoprecipitation of the purified hFasLECDs. a) Co-immunoprecipitation using wild-type hFasRECD-T-Fc [ 15 ]. Lanes: M, molecular-weight size-markers; a, NFG5-hFasLECD [ 10 ]; b, tag-free hFasLECD; c, N -Ethylmaleimide adduct of NFG1CG4-hFasLECD, d, SUNBRIGHT® ME-050MA adduct of NFG1CG4-hFasLECD; e, buffer alone. The bands in “FasR-Fc” labeled region were derived from wild-type hFasRECD-T-Fc sample. b) Comparison between wild-type hFasRECD-T-Fc (lanes a, c and e) and hFasRECD-T-Fc (N102Q, N120Q) mutant (lanes b, d and f). Lanes: M, molecular-weight size-markers; a and b, tag-free hFasLECD; c and d, N -Ethylmaleimide adduct of NFG1CG4-hFasLECD; e and f, SUNBRIGHT® ME-050MA adduct of NFG1CG4-hFasLECD. The bands in “FasR-Fc” labeled region were derived from hFasRECD-T-Fc samples.
    Figure Legend Snippet: SDS-PAGE analysis of hFasRECD-T-Fc mediated co-immunoprecipitation of the purified hFasLECDs. a) Co-immunoprecipitation using wild-type hFasRECD-T-Fc [ 15 ]. Lanes: M, molecular-weight size-markers; a, NFG5-hFasLECD [ 10 ]; b, tag-free hFasLECD; c, N -Ethylmaleimide adduct of NFG1CG4-hFasLECD, d, SUNBRIGHT® ME-050MA adduct of NFG1CG4-hFasLECD; e, buffer alone. The bands in “FasR-Fc” labeled region were derived from wild-type hFasRECD-T-Fc sample. b) Comparison between wild-type hFasRECD-T-Fc (lanes a, c and e) and hFasRECD-T-Fc (N102Q, N120Q) mutant (lanes b, d and f). Lanes: M, molecular-weight size-markers; a and b, tag-free hFasLECD; c and d, N -Ethylmaleimide adduct of NFG1CG4-hFasLECD; e and f, SUNBRIGHT® ME-050MA adduct of NFG1CG4-hFasLECD. The bands in “FasR-Fc” labeled region were derived from hFasRECD-T-Fc samples.

    Techniques Used: SDS Page, Immunoprecipitation, Purification, Molecular Weight, Labeling, Derivative Assay, Mutagenesis

    4) Product Images from "16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis"

    Article Title: 16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis

    Journal: Endocrine Connections

    doi: 10.1530/EC-18-0116

    Vasoinhibin binds integrin alpha5 beta1 rVi and mPRL after immunoprecipitation of integrin alpha5 beta1 were detected by western blot using an anti-prolactin antibody. Mouse IgG was used for immunoprecipitation as a negative control. rVi was detected after immunoprecipitation, but mPRL was not detected.
    Figure Legend Snippet: Vasoinhibin binds integrin alpha5 beta1 rVi and mPRL after immunoprecipitation of integrin alpha5 beta1 were detected by western blot using an anti-prolactin antibody. Mouse IgG was used for immunoprecipitation as a negative control. rVi was detected after immunoprecipitation, but mPRL was not detected.

    Techniques Used: Immunoprecipitation, Western Blot, Negative Control

    5) Product Images from "Recoding RNA editing of antizyme inhibitor 1 predisposes to hepatocellular carcinoma"

    Article Title: Recoding RNA editing of antizyme inhibitor 1 predisposes to hepatocellular carcinoma

    Journal: Nature medicine

    doi: 10.1038/nm.3043

    Edited AZIN1 neutralizes antizyme-mediated degradation of target oncoproteins in vitro and in vivo . ( a ) Co-immunoprecipitation of antizyme-1 in PLC8024 cells transfected with the indicated constructs using a GFP-specific antibody (IP: GFP) or mouse IgG
    Figure Legend Snippet: Edited AZIN1 neutralizes antizyme-mediated degradation of target oncoproteins in vitro and in vivo . ( a ) Co-immunoprecipitation of antizyme-1 in PLC8024 cells transfected with the indicated constructs using a GFP-specific antibody (IP: GFP) or mouse IgG

    Techniques Used: In Vitro, In Vivo, Immunoprecipitation, Transfection, Construct

    6) Product Images from "A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR"

    Article Title: A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14606-3

    The effects of SLC26A3-p.Asp688His mutation on SLC26A3-CFTR interaction and regulation. COS7 cells were transfected or co-transfected either with the HA-tagged CFTR R domain (HA-RD), with the Myc-tagged wild-type SLC26A3 STAS domain (Myc-STAS), with the Myc-tagged SLC26A3 STAS domain with the c.2062 G > C (p.Asp688His) mutation (Myc-STAS-p.Asp688His), or with an empty HA-vector, as indicated in the corresponding lanes. Cell lysates were used for western blotting ( A ) and immunoprecipitation ( B ). Peptides of correct sizes were detected in the immunoblots by using the anti-Myc, anti-HA, or anti-GAPDH antibodies, as indicated. Immunostaining of GAPDH served as a loading control. In the immunoprecipitation experiments ( B ), cells were co-transfected with CFTR HA-RD and with Myc-STAS or Myc-STAS-p.Asp688His. Anti-HA-antibody or normal rabbit IgG (negative control) were used for the precipitation, and anti-Myc-antibody was used for detection on the blot. Extracts show co-immunoprecipitation of the HA-RD with both the wild-type (Myc-STAS) and the mutant p.Asp688His (Myc-STAS-p.Asp688His) STAS domains. ( C ) Representative traces and ( D ) summary of the current mediated by CFTR-dependent Cl − transport in Xenopus oocytes expressing CFTR alone or co-expressing CFTR with wild-type (WT) SLC26A3, SLC26A3-p.Asp688His or both. The numbers in the columns indicate the number of experiments for each condition. ( E ) CFTR trafficking to the plasma membrane monitored by using a biotinylation assay in HEK293 cells expressing CFTR, wild-type SLC26A3, or SLC26A3-p.Asp688His as indicated.
    Figure Legend Snippet: The effects of SLC26A3-p.Asp688His mutation on SLC26A3-CFTR interaction and regulation. COS7 cells were transfected or co-transfected either with the HA-tagged CFTR R domain (HA-RD), with the Myc-tagged wild-type SLC26A3 STAS domain (Myc-STAS), with the Myc-tagged SLC26A3 STAS domain with the c.2062 G > C (p.Asp688His) mutation (Myc-STAS-p.Asp688His), or with an empty HA-vector, as indicated in the corresponding lanes. Cell lysates were used for western blotting ( A ) and immunoprecipitation ( B ). Peptides of correct sizes were detected in the immunoblots by using the anti-Myc, anti-HA, or anti-GAPDH antibodies, as indicated. Immunostaining of GAPDH served as a loading control. In the immunoprecipitation experiments ( B ), cells were co-transfected with CFTR HA-RD and with Myc-STAS or Myc-STAS-p.Asp688His. Anti-HA-antibody or normal rabbit IgG (negative control) were used for the precipitation, and anti-Myc-antibody was used for detection on the blot. Extracts show co-immunoprecipitation of the HA-RD with both the wild-type (Myc-STAS) and the mutant p.Asp688His (Myc-STAS-p.Asp688His) STAS domains. ( C ) Representative traces and ( D ) summary of the current mediated by CFTR-dependent Cl − transport in Xenopus oocytes expressing CFTR alone or co-expressing CFTR with wild-type (WT) SLC26A3, SLC26A3-p.Asp688His or both. The numbers in the columns indicate the number of experiments for each condition. ( E ) CFTR trafficking to the plasma membrane monitored by using a biotinylation assay in HEK293 cells expressing CFTR, wild-type SLC26A3, or SLC26A3-p.Asp688His as indicated.

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Immunostaining, Negative Control, Expressing, Cell Surface Biotinylation Assay

    7) Product Images from "Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells"

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00418.2008

    Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).
    Figure Legend Snippet: Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).

    Techniques Used: Western Blot, Expressing, Immunoprecipitation

    8) Product Images from "Loss of renal SNX5 results in impaired IDE activity and insulin resistance in mice"

    Article Title: Loss of renal SNX5 results in impaired IDE activity and insulin resistance in mice

    Journal: Diabetologia

    doi: 10.1007/s00125-017-4482-1

    Co-localisation and co-immunoprecipitation of IDE and SNX5 in hRPTCs. (a) Co-localisation of IDE and SNX5 in hRPTCs from normotensive human volunteers. The cells were washed, fixed and immunostained for IDE and SNX5. Co-localisation is visualised as yellow discrete areas in merged images of IDE (pseudocoloured green) and SNX5 (pseudocoloured red). There is co-localisation at the plasma membrane and perinuclear area in the basal state. Insulin treatment (100 nmol/l, 30 min) increased the co-localisation at the plasma membrane and perinuclear area; scale bar, 5 μm. (b). FRET between IDE and SNX5 in hRPTCs. Alexa Fluor 488-labelled IDE was used as the donor and Alexa Fluor 555-labelled SNX5 was used as the acceptor in the FRET dipole. At 30 min, the FRET between IDE and SNX5 had an energy transfer efficiency (E%) around 50%. uFRET, uncorrected FRET; pFRET, processed FRET. (c) Co-immunoprecipitation of IDE and SNX5 in hRPTCs. The cells were immunoprecipitated (IP) with SNX5 antibodies and immunoblotted for IDE. The co-immunoprecipitation of IDE and SNX5 occurred following insulin treatment (100 nmol/l, 30 min). NC, negative control with non-specific IgG; PC, positive control with regular immunoblotting for IDE of the cell lysates
    Figure Legend Snippet: Co-localisation and co-immunoprecipitation of IDE and SNX5 in hRPTCs. (a) Co-localisation of IDE and SNX5 in hRPTCs from normotensive human volunteers. The cells were washed, fixed and immunostained for IDE and SNX5. Co-localisation is visualised as yellow discrete areas in merged images of IDE (pseudocoloured green) and SNX5 (pseudocoloured red). There is co-localisation at the plasma membrane and perinuclear area in the basal state. Insulin treatment (100 nmol/l, 30 min) increased the co-localisation at the plasma membrane and perinuclear area; scale bar, 5 μm. (b). FRET between IDE and SNX5 in hRPTCs. Alexa Fluor 488-labelled IDE was used as the donor and Alexa Fluor 555-labelled SNX5 was used as the acceptor in the FRET dipole. At 30 min, the FRET between IDE and SNX5 had an energy transfer efficiency (E%) around 50%. uFRET, uncorrected FRET; pFRET, processed FRET. (c) Co-immunoprecipitation of IDE and SNX5 in hRPTCs. The cells were immunoprecipitated (IP) with SNX5 antibodies and immunoblotted for IDE. The co-immunoprecipitation of IDE and SNX5 occurred following insulin treatment (100 nmol/l, 30 min). NC, negative control with non-specific IgG; PC, positive control with regular immunoblotting for IDE of the cell lysates

    Techniques Used: Immunoprecipitation, Negative Control, Positive Control

    9) Product Images from "Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex"

    Article Title: Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052640

    Schematic overview of the SILAC approach. “Heavy”-labeled cells were co-transfected with the plasmids CHD7-CR1-3-pCMV-HA (containing amino acids 1593-2178, NP_060250.2, in fusion with an HA-tag) and CHD8-pCMV-cmyc (spanning amino acids 1789–2302, NP_065971.2, in fusion with a cmyc-tag). The CHD7 part was purified by anti-HA immunoprecipitation. As a negative control, the same immunoprecipitation was performed in lysates of non-transfected “Light”-labeled HeLa cells. Purified proteins from both cell cultures were pooled in equimolar amounts and in-gel digested, followed by liquid-chromatography (LC)-coupled tandem mass spectrometry.
    Figure Legend Snippet: Schematic overview of the SILAC approach. “Heavy”-labeled cells were co-transfected with the plasmids CHD7-CR1-3-pCMV-HA (containing amino acids 1593-2178, NP_060250.2, in fusion with an HA-tag) and CHD8-pCMV-cmyc (spanning amino acids 1789–2302, NP_065971.2, in fusion with a cmyc-tag). The CHD7 part was purified by anti-HA immunoprecipitation. As a negative control, the same immunoprecipitation was performed in lysates of non-transfected “Light”-labeled HeLa cells. Purified proteins from both cell cultures were pooled in equimolar amounts and in-gel digested, followed by liquid-chromatography (LC)-coupled tandem mass spectrometry.

    Techniques Used: Labeling, Transfection, Purification, Immunoprecipitation, Negative Control, Liquid Chromatography, Mass Spectrometry

    Co-immunoprecipitation of FAM124B with a part of CHD7 and CHD8. HeLa cells were co-transfected with either the CHD7-CR1-3-pCMV-HA (amino acids 1593-2178, NP_060250.2) plasmid and FAM124B-1,3-pCMV-cmyc or FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1) or with CHD8-pCMV-cmyc (amino acids 1789-2302, NP_065971.2) and FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1). ( A ) Using the anti-CHD8 (abcam, ab84527) or the anti-CHD7 (abcam, ab31824) antibody for precipitation, we detected with the anti-HA antibody (Roche) an approximately 51 kDa band corresponding to the estimated size of FAM124B transcript variant 1. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( B ) Reciprocal immunoprecipitation with anti-cmyc antibody (precipitating FAM124B transcript variant 1), and detection with the anti-CHD7 antibody lead to a specific band ∼70 kDa, the estimated size for the CHD7 part fused to the HA-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( C ) Reciprocal experiment with anti-HA antibody (precipitating FAM124B transcript variant 1) and detection with the anti-CHD8 antibody detected a specific band ∼68 kDa, the estimated size for the CHD8 part fused to the cmyc-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( D, E, F ) The same experimental procedure was performed for FAM124B transcript variant 2, demonstrating a specific interaction of FAM124B transcript variant 2 with the CHD7 and CHD8 part as well. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control.
    Figure Legend Snippet: Co-immunoprecipitation of FAM124B with a part of CHD7 and CHD8. HeLa cells were co-transfected with either the CHD7-CR1-3-pCMV-HA (amino acids 1593-2178, NP_060250.2) plasmid and FAM124B-1,3-pCMV-cmyc or FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1) or with CHD8-pCMV-cmyc (amino acids 1789-2302, NP_065971.2) and FAM124B-1,3-pCMV-HA (transcript variant 1, NP_001116251.1). ( A ) Using the anti-CHD8 (abcam, ab84527) or the anti-CHD7 (abcam, ab31824) antibody for precipitation, we detected with the anti-HA antibody (Roche) an approximately 51 kDa band corresponding to the estimated size of FAM124B transcript variant 1. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( B ) Reciprocal immunoprecipitation with anti-cmyc antibody (precipitating FAM124B transcript variant 1), and detection with the anti-CHD7 antibody lead to a specific band ∼70 kDa, the estimated size for the CHD7 part fused to the HA-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( C ) Reciprocal experiment with anti-HA antibody (precipitating FAM124B transcript variant 1) and detection with the anti-CHD8 antibody detected a specific band ∼68 kDa, the estimated size for the CHD8 part fused to the cmyc-tag. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control. ( D, E, F ) The same experimental procedure was performed for FAM124B transcript variant 2, demonstrating a specific interaction of FAM124B transcript variant 2 with the CHD7 and CHD8 part as well. Lane 1: co-transfected Co-IP, lane 2: untransfected HeLa cells as negative control.

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Variant Assay, Co-Immunoprecipitation Assay, Negative Control

    10) Product Images from "Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation"

    Article Title: Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation

    Journal:

    doi: 10.1073/pnas.0406789102

    Interaction and ubiquitination of FOXO1 by Skp2 depend on phosphorylation of FOXO1 at Ser-256. ( A ) FOXO1 plasmids were cotransfected with Skp2 into LNCaP cells, and cell extracts were prepared for immunoprecipitation with an anti-FLAG antibody and immunoblotted
    Figure Legend Snippet: Interaction and ubiquitination of FOXO1 by Skp2 depend on phosphorylation of FOXO1 at Ser-256. ( A ) FOXO1 plasmids were cotransfected with Skp2 into LNCaP cells, and cell extracts were prepared for immunoprecipitation with an anti-FLAG antibody and immunoblotted

    Techniques Used: Immunoprecipitation

    11) Product Images from "16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis"

    Article Title: 16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis

    Journal: Endocrine Connections

    doi: 10.1530/EC-18-0116

    Vasoinhibin binds integrin alpha5 beta1 rVi and mPRL after immunoprecipitation of integrin alpha5 beta1 were detected by western blot using an anti-prolactin antibody. Mouse IgG was used for immunoprecipitation as a negative control. rVi was detected after immunoprecipitation, but mPRL was not detected.
    Figure Legend Snippet: Vasoinhibin binds integrin alpha5 beta1 rVi and mPRL after immunoprecipitation of integrin alpha5 beta1 were detected by western blot using an anti-prolactin antibody. Mouse IgG was used for immunoprecipitation as a negative control. rVi was detected after immunoprecipitation, but mPRL was not detected.

    Techniques Used: Immunoprecipitation, Western Blot, Negative Control

    12) Product Images from "PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription"

    Article Title: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-2-5

    PNRC associates with RPC39 in mammalian cells . MCF-7/EGFP or MCF7/EGFP-PNRC stable expression cells were transiently transfected with the RPC39 expression plasmid, pSG5-RPC39, 24 h post-transfection, cells were harvasted and lysed, and 15 mg of total proteins were immunoprecipitated with antibodies against GFP (Clontech) or RPC39 (Santa Cruz Biotechnology). Specifically bound proteins to Protein A agarose beads were separated on a 10% SDS-PAGE and analysed by immunoblotting with either anti-RPC39 ( A , blot: anti-RPC39) or anti-GFP ( B , blot: anti-GFP) antibody, as previously described [2]. An aliquot of cell lysate equal to 100 μg of protein was included in each SDS-PAGE gel for Western blot to examine whether the crude protein extracts used for co-immunoprecipitation contains EGFP-PNRC and RPC39 proteins. The protein bands with molecular weights corresponding to those of RPC39 or EGFP-PNRC were indicated by arrows.
    Figure Legend Snippet: PNRC associates with RPC39 in mammalian cells . MCF-7/EGFP or MCF7/EGFP-PNRC stable expression cells were transiently transfected with the RPC39 expression plasmid, pSG5-RPC39, 24 h post-transfection, cells were harvasted and lysed, and 15 mg of total proteins were immunoprecipitated with antibodies against GFP (Clontech) or RPC39 (Santa Cruz Biotechnology). Specifically bound proteins to Protein A agarose beads were separated on a 10% SDS-PAGE and analysed by immunoblotting with either anti-RPC39 ( A , blot: anti-RPC39) or anti-GFP ( B , blot: anti-GFP) antibody, as previously described [2]. An aliquot of cell lysate equal to 100 μg of protein was included in each SDS-PAGE gel for Western blot to examine whether the crude protein extracts used for co-immunoprecipitation contains EGFP-PNRC and RPC39 proteins. The protein bands with molecular weights corresponding to those of RPC39 or EGFP-PNRC were indicated by arrows.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot

    13) Product Images from "Human Metapneumovirus Glycoprotein G Disrupts Mitochondrial Signaling in Airway Epithelial Cells"

    Article Title: Human Metapneumovirus Glycoprotein G Disrupts Mitochondrial Signaling in Airway Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062568

    G protein blocks the interaction between RIG-I and MAVS. ( A ) 293 cells were transfected with plasmids encoding Flag-tagged RIG-I-N and V5-tagged G or their control vectors. Total cell lysates were immunoprecipitated with anti-V5 antibody followed by Western blot using anti-Flag antibody to detect RIG-I-N. Reverse immunoprecipitation was also done, where RIG-I-N was immunoprecipitated using anti-Flag antibody and G protein was then detected using anti-V5 antibody. Total cell lysates were subjected to Western blot to determine levels of hMPV G and RIG-I-N expression. ( B ) 293 cells were transfected with a fixed amount of plasmids encoding Flag-tagged RIG-I and EGFP-tagged MAVS, or their control vectors, and increasing concentrations of a plasmid expressing V5-tagged G. Total cell lysates were immunoprecipitated with anti-Flag antibody to pull down RIG-I, followed by Western blot using anti-EGFP or anti-V5 antibody to detect associated MAVS and G, respectively. Data are representative of two independent experiments.
    Figure Legend Snippet: G protein blocks the interaction between RIG-I and MAVS. ( A ) 293 cells were transfected with plasmids encoding Flag-tagged RIG-I-N and V5-tagged G or their control vectors. Total cell lysates were immunoprecipitated with anti-V5 antibody followed by Western blot using anti-Flag antibody to detect RIG-I-N. Reverse immunoprecipitation was also done, where RIG-I-N was immunoprecipitated using anti-Flag antibody and G protein was then detected using anti-V5 antibody. Total cell lysates were subjected to Western blot to determine levels of hMPV G and RIG-I-N expression. ( B ) 293 cells were transfected with a fixed amount of plasmids encoding Flag-tagged RIG-I and EGFP-tagged MAVS, or their control vectors, and increasing concentrations of a plasmid expressing V5-tagged G. Total cell lysates were immunoprecipitated with anti-Flag antibody to pull down RIG-I, followed by Western blot using anti-EGFP or anti-V5 antibody to detect associated MAVS and G, respectively. Data are representative of two independent experiments.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation

    14) Product Images from "Soybean ENOD40 encodes two peptides that bind to sucrose synthase"

    Article Title: Soybean ENOD40 encodes two peptides that bind to sucrose synthase

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

    doi: 10.1073/pnas.022664799

    Immunoprecipitation of in vitro translation products. [ 35 S]-Methionine-labeled peptides translated in wheat germ extracts from wild-type GmENOD40 mRNA were solid-phase extracted (lane 1, control), immunoprecipitated with antigen-specific IgGs (lanes 2 and 3), and analyzed by Tricine-SDS/16.5% PAGE and autoradiography. For immunoprecipitation, affinity-purified Abs directed against ORF A- and ORF B-deduced synthetic peptides were used.
    Figure Legend Snippet: Immunoprecipitation of in vitro translation products. [ 35 S]-Methionine-labeled peptides translated in wheat germ extracts from wild-type GmENOD40 mRNA were solid-phase extracted (lane 1, control), immunoprecipitated with antigen-specific IgGs (lanes 2 and 3), and analyzed by Tricine-SDS/16.5% PAGE and autoradiography. For immunoprecipitation, affinity-purified Abs directed against ORF A- and ORF B-deduced synthetic peptides were used.

    Techniques Used: Immunoprecipitation, In Vitro, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Affinity Purification

    15) Product Images from "Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells"

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00418.2008

    Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).
    Figure Legend Snippet: Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).

    Techniques Used: Western Blot, Expressing, Immunoprecipitation

    16) Product Images from "Targeted Deletion of Tssk1 2 Causes Male Infertility Due to Haploinsufficiency"

    Article Title: Targeted Deletion of Tssk1 2 Causes Male Infertility Due to Haploinsufficiency

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2008.03.047

    Co-immunoprecipitation and in vitro phosphorylation of TSKS/TSSK2 complexes from mouse testis In panels 1 and 2, pre-cleared mouse testis extracts were immunoprecipitated with either rabbit normal serum (Rabbit IP-control), or rabbit anti-TSSK2 serum (IP-TSSK2), and the immunoprecipitates were subjected to immunoblotting with anti-TSSK2 (panel 1) or anti-TSKS (panel 2). TSKS and TSSK2 were both detected in the immune-complex immunoprecipitated with anti-TSSK2 serum. In panels 3 and 4, precleared mouse testis extracts were immunoprecipitated with either normal rat serum (Rat IP-control), or rat anti-TSKS serum (IP-TSKS), and the immunoprecipitated complexes were subjected to immunoblotting with anti-TSKS (panel 3) or anti-TSSK2 antibody (panel 4). Both TSKS and TSSK2 were detected in the immune-complex immunoprecipitated with anti-TSKS serum. Panel 5 shows an autoradiograph of immune-complexes (as described in panel 3 and 4) immunoprecipitated with either anti-TSKS (IP-TSKS) or rat normal serum (Rat IP-control) and subsequently incubated with [ 32 P]γATP in an in vitro kinase assay. A common kinase, casein kinase 2 (CKII) and several common kinase substrates including casein, myelin basic protein (MBP) and histone 3 were added to the reactions as the positive control. In immunoprecipitates with anti-TSKS sera, TSKS was strongly phosphorylated as well as a ~40 kDa protein, while no proteins were phosphorylated in immunoprecipitates with control normal rat sera. The ~40 kDa protein may represent autophosphorylation of TSSK1 and/or TSSK2 or the truncated form of TSKS. Addition of CKII did not result in any additional phosphorylation. Casein, MBP and histone3 were also phosphorylated by the kinases in the precipitated complex, whereas these proteins were not phosphorylated in the controls immunoprecipitated by normal sera.
    Figure Legend Snippet: Co-immunoprecipitation and in vitro phosphorylation of TSKS/TSSK2 complexes from mouse testis In panels 1 and 2, pre-cleared mouse testis extracts were immunoprecipitated with either rabbit normal serum (Rabbit IP-control), or rabbit anti-TSSK2 serum (IP-TSSK2), and the immunoprecipitates were subjected to immunoblotting with anti-TSSK2 (panel 1) or anti-TSKS (panel 2). TSKS and TSSK2 were both detected in the immune-complex immunoprecipitated with anti-TSSK2 serum. In panels 3 and 4, precleared mouse testis extracts were immunoprecipitated with either normal rat serum (Rat IP-control), or rat anti-TSKS serum (IP-TSKS), and the immunoprecipitated complexes were subjected to immunoblotting with anti-TSKS (panel 3) or anti-TSSK2 antibody (panel 4). Both TSKS and TSSK2 were detected in the immune-complex immunoprecipitated with anti-TSKS serum. Panel 5 shows an autoradiograph of immune-complexes (as described in panel 3 and 4) immunoprecipitated with either anti-TSKS (IP-TSKS) or rat normal serum (Rat IP-control) and subsequently incubated with [ 32 P]γATP in an in vitro kinase assay. A common kinase, casein kinase 2 (CKII) and several common kinase substrates including casein, myelin basic protein (MBP) and histone 3 were added to the reactions as the positive control. In immunoprecipitates with anti-TSKS sera, TSKS was strongly phosphorylated as well as a ~40 kDa protein, while no proteins were phosphorylated in immunoprecipitates with control normal rat sera. The ~40 kDa protein may represent autophosphorylation of TSSK1 and/or TSSK2 or the truncated form of TSKS. Addition of CKII did not result in any additional phosphorylation. Casein, MBP and histone3 were also phosphorylated by the kinases in the precipitated complex, whereas these proteins were not phosphorylated in the controls immunoprecipitated by normal sera.

    Techniques Used: Immunoprecipitation, In Vitro, Autoradiography, Incubation, Kinase Assay, Positive Control

    Co-immunoprecipitation and in vitro phosphorylation of TSKS/TSSK2 complexes from human sperm Panels 1 and 2: pre-cleared human sperm extracts were immunoprecipitated with either control rabbit serum (Rabbit IP-control) or rabbit anti-TSSK2 serum (IP-TSSK2), and the immunoprecipitates were subjected to immunoblotting with anti-TSSK2 (panel 1) or anti-TSKS (panel 2). 65 kDa and ~50 kDa TSKS isoforms were recognized by anti-TSKS antibody only in the lane immunoprecipitated with anti-TSSK2 antibody while 42 kDa TSSK2 was detected in the immunoprecipitate. Panels 5 and 6: pre-cleared human sperm extracts were immunoprecipitated with either pre-immune rat serum (Rat IP-control), or rat anti-TSKS serum (IP-TSKS), the immunoprecipitates were subjected to immunoblotting with anti-TSKS antibody (panel 5) or anti-TSSK2 antibody (panel 6). 65 kDa TSKS and 42 kDa TSSK2 were detected only in the lanes immunoprecipitated with anti-TSKS. Panels 3 and 4: autoradiograph of TSSK2/TSKS complexes immunoprecipitated with either anti-TSSK2 (IP-TSSK2) or anti-TSKS (IP-TSKS) and incubated with [ 32 P]γATP. Four identical phospho-protein bands were detected in immune complexs precipitated with either anti-TSSK2 (IP-TSSK2) or anti-TSKS (IP-TSKS). Conversely, no proteins were phosphorylated in the controls immunoprecipitated with normal sera (Rabbit IP-control and Rat IP-control). These bands represent phosphorylation of 65 and ~50 kDa forms of TSKS, autophosphorylation of TSSK2 and an unknown 30 kDa phosphoprotein.
    Figure Legend Snippet: Co-immunoprecipitation and in vitro phosphorylation of TSKS/TSSK2 complexes from human sperm Panels 1 and 2: pre-cleared human sperm extracts were immunoprecipitated with either control rabbit serum (Rabbit IP-control) or rabbit anti-TSSK2 serum (IP-TSSK2), and the immunoprecipitates were subjected to immunoblotting with anti-TSSK2 (panel 1) or anti-TSKS (panel 2). 65 kDa and ~50 kDa TSKS isoforms were recognized by anti-TSKS antibody only in the lane immunoprecipitated with anti-TSSK2 antibody while 42 kDa TSSK2 was detected in the immunoprecipitate. Panels 5 and 6: pre-cleared human sperm extracts were immunoprecipitated with either pre-immune rat serum (Rat IP-control), or rat anti-TSKS serum (IP-TSKS), the immunoprecipitates were subjected to immunoblotting with anti-TSKS antibody (panel 5) or anti-TSSK2 antibody (panel 6). 65 kDa TSKS and 42 kDa TSSK2 were detected only in the lanes immunoprecipitated with anti-TSKS. Panels 3 and 4: autoradiograph of TSSK2/TSKS complexes immunoprecipitated with either anti-TSSK2 (IP-TSSK2) or anti-TSKS (IP-TSKS) and incubated with [ 32 P]γATP. Four identical phospho-protein bands were detected in immune complexs precipitated with either anti-TSSK2 (IP-TSSK2) or anti-TSKS (IP-TSKS). Conversely, no proteins were phosphorylated in the controls immunoprecipitated with normal sera (Rabbit IP-control and Rat IP-control). These bands represent phosphorylation of 65 and ~50 kDa forms of TSKS, autophosphorylation of TSSK2 and an unknown 30 kDa phosphoprotein.

    Techniques Used: Immunoprecipitation, In Vitro, Autoradiography, Incubation

    17) Product Images from "Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair"

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0263-x

    Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p
    Figure Legend Snippet: Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p

    Techniques Used: Immunoprecipitation, Expressing, Immunofluorescence, Staining, Western Blot, Negative Control

    18) Product Images from "Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation"

    Article Title: Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061760

    Interactions beetween adhesion proteins during myoblast differentiation in vitro . A - colocalization of integrin alpha3 (green) and integrin beta1, ADAM12, CD9, CD81, M-cadherin, or VCAM-1 (red). Scale bars 50 µm. Arrows show the area where colocalization was not observed. B - Immunoprecipitation of integrin alpha3 and subsequent Western blotting analysis of CD9, CD81, M-cadherin, and V-CAM-1, c – control immunoblotting with whole cell lysate. C - interactions of analysed adhesion proteis during myoblasts differentiation. The table sumarize current study and previous results [38] .
    Figure Legend Snippet: Interactions beetween adhesion proteins during myoblast differentiation in vitro . A - colocalization of integrin alpha3 (green) and integrin beta1, ADAM12, CD9, CD81, M-cadherin, or VCAM-1 (red). Scale bars 50 µm. Arrows show the area where colocalization was not observed. B - Immunoprecipitation of integrin alpha3 and subsequent Western blotting analysis of CD9, CD81, M-cadherin, and V-CAM-1, c – control immunoblotting with whole cell lysate. C - interactions of analysed adhesion proteis during myoblasts differentiation. The table sumarize current study and previous results [38] .

    Techniques Used: In Vitro, Immunoprecipitation, Western Blot, Chick Chorioallantoic Membrane Assay

    19) Product Images from "Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair"

    Article Title: Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-2

    The interaction of DNA-PKcs with cyclin T2 . A: Coomassie bright blue dye staining of SDS-PAGE of the co-immunoprecipitation products of DNA-PKcs antibody. B: Immunoblotting analysis of cyclin T2 was performed on the CoIP products of DNA-PKcs antibody and total extracts of cells. Three DNA-PKcs lanes represent three independent repeat IP experiments. C: Immunoblotting analysis of DNA-PKcs and cyclin T2 was performed on the CoIP product of cyclin T2 antibody and the total extracts of cells. The immunoprecipitation product of Ig G was taken as the blank control.
    Figure Legend Snippet: The interaction of DNA-PKcs with cyclin T2 . A: Coomassie bright blue dye staining of SDS-PAGE of the co-immunoprecipitation products of DNA-PKcs antibody. B: Immunoblotting analysis of cyclin T2 was performed on the CoIP products of DNA-PKcs antibody and total extracts of cells. Three DNA-PKcs lanes represent three independent repeat IP experiments. C: Immunoblotting analysis of DNA-PKcs and cyclin T2 was performed on the CoIP product of cyclin T2 antibody and the total extracts of cells. The immunoprecipitation product of Ig G was taken as the blank control.

    Techniques Used: Staining, SDS Page, Immunoprecipitation, Co-Immunoprecipitation Assay

    20) Product Images from "Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney"

    Article Title: Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney

    Journal: Bioscience Reports

    doi: 10.1042/BSR20170163

    ODC and OAZ co-immunoprecipitation and visualization by immunoblotting Cos-7 cells were transfected with cDNAs encoding tagged proteins. Panel ( A ) shows proteins immunoprecipitated with mouse anti-Myc antibody and immunoblotted with rabbit anti-FLAG antibody. The left side in panel (A) shows a negative control with only FLAG-tagged proteins. Although the negative controls should be clear, faint ODCrp and Δ1–13 ODCrp bands can be seen due to unspecific binding. The fact that the bands are equally faint in the co-immunoprecipitation samples (right side in panel (A)) shows that ODCrp does not co-precipitate with ODC or OAZ, indicating that ODCrp does not form stable heterodimers. Panel ( B ) shows pre-immunoprecipitation samples probed with anti-FLAG (upper) and anti-Myc (lower) antibodies to verify the presence of transfected proteins. The samples in panel (B) are in the same order as in panel (A), except for the positive ODC – ODC control, which is replaced with marker.
    Figure Legend Snippet: ODC and OAZ co-immunoprecipitation and visualization by immunoblotting Cos-7 cells were transfected with cDNAs encoding tagged proteins. Panel ( A ) shows proteins immunoprecipitated with mouse anti-Myc antibody and immunoblotted with rabbit anti-FLAG antibody. The left side in panel (A) shows a negative control with only FLAG-tagged proteins. Although the negative controls should be clear, faint ODCrp and Δ1–13 ODCrp bands can be seen due to unspecific binding. The fact that the bands are equally faint in the co-immunoprecipitation samples (right side in panel (A)) shows that ODCrp does not co-precipitate with ODC or OAZ, indicating that ODCrp does not form stable heterodimers. Panel ( B ) shows pre-immunoprecipitation samples probed with anti-FLAG (upper) and anti-Myc (lower) antibodies to verify the presence of transfected proteins. The samples in panel (B) are in the same order as in panel (A), except for the positive ODC – ODC control, which is replaced with marker.

    Techniques Used: Immunoprecipitation, Transfection, Negative Control, Binding Assay, Marker

    ODC and OAZ co-immunoprecipitation and visualization by immunoblotting. Panel A and B show reciprocal experiment of that represented in Fig. 6 in main text. Cos-7 cells were transfected with cDNAs encoding tagged proteins. To the right of the marker (M) are proteins immunoprecipitated with mouse anti-FLAG antibody and immunoblotted with rabbit anti-Myc antibody. Pre-immunoprecipitation samples are to the left of the marker. Pre-immunoprecipitation samples were immunoblotted with both anti-FLAG and anti-Myc antibodies to verify the presence of transfected proteins. In panel A , only ODC-FLAG co-precipitated with ODC-Myc, indicating that ODCrp and Δ1–13 ODCrp do not form stable dimers with ODC. In panel B , only AZIN1-FLAG co-precipitated with OAZ-Myc, verifying that ODCrp and Δ1–13 ODCrp do not form stable dimers with OAZ.
    Figure Legend Snippet: ODC and OAZ co-immunoprecipitation and visualization by immunoblotting. Panel A and B show reciprocal experiment of that represented in Fig. 6 in main text. Cos-7 cells were transfected with cDNAs encoding tagged proteins. To the right of the marker (M) are proteins immunoprecipitated with mouse anti-FLAG antibody and immunoblotted with rabbit anti-Myc antibody. Pre-immunoprecipitation samples are to the left of the marker. Pre-immunoprecipitation samples were immunoblotted with both anti-FLAG and anti-Myc antibodies to verify the presence of transfected proteins. In panel A , only ODC-FLAG co-precipitated with ODC-Myc, indicating that ODCrp and Δ1–13 ODCrp do not form stable dimers with ODC. In panel B , only AZIN1-FLAG co-precipitated with OAZ-Myc, verifying that ODCrp and Δ1–13 ODCrp do not form stable dimers with OAZ.

    Techniques Used: Immunoprecipitation, Transfection, Marker

    21) Product Images from "Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development"

    Article Title: Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

    Journal: Infection and Immunity

    doi: 10.1128/IAI.69.10.6475-6482.2001

    Characterization of MAb 11D5/H3 by indirect immunofluorescence and immunoprecipitation of the target antigen. Bars, 10 μm. (A) Staining pattern of air-dried sporozoites when the antibody was applied after the permeabilization of parasites with acetone. (B and C) Phase-contrast image and corresponding immunofluorescence of L2 pneumonocytes ethanol-acetone-fixed 1 h postinfection with sporozoites. Sporozoites were preincubated with mouse MAb 11D5/H3 before inoculation onto cells. After fixation and permeabilization of cells, 11D5/H3 was visualized with FITC-conjugated anti-mouse IgG. An extracellular sporozoite (arrow; above the focus level of host cell cy- toplasm) is labeled on the surface (inset shows a magnification) as indicated by the patchiness of membrane staining, whereas the label is absent on an intracellular sporozoite (arrowhead). The latter resides inside a PV which is visible by the surrounding halo. (D) Electrophoretic separation of the antigen precipitated by MAb 11D5/H3 from sporocyst-sporozoite extracts in gel-loading buffer containing 4% 2-mercaptoethanol (lane 2). A major band at 58 kDa was detected, whereby two minor bands appeared at 27 and 31 kDa. MAb 11D5/H3 did not react with bradyzoite extracts (lane 1), indicating that the antigen was not expressed in bradyzoites. An irrelevant isotype-matched MAb (IgG2a) and a sporozoite-specific rabbit serum (K3) served as negative and positive controls, respectively. (E) Electrophoretic separation of the antigen precipitated by 11D5/H3 from sporocyst-sporozoite extracts as performed before, except that reducing conditions were lowered to 2% 2-mercaptoethanol.
    Figure Legend Snippet: Characterization of MAb 11D5/H3 by indirect immunofluorescence and immunoprecipitation of the target antigen. Bars, 10 μm. (A) Staining pattern of air-dried sporozoites when the antibody was applied after the permeabilization of parasites with acetone. (B and C) Phase-contrast image and corresponding immunofluorescence of L2 pneumonocytes ethanol-acetone-fixed 1 h postinfection with sporozoites. Sporozoites were preincubated with mouse MAb 11D5/H3 before inoculation onto cells. After fixation and permeabilization of cells, 11D5/H3 was visualized with FITC-conjugated anti-mouse IgG. An extracellular sporozoite (arrow; above the focus level of host cell cy- toplasm) is labeled on the surface (inset shows a magnification) as indicated by the patchiness of membrane staining, whereas the label is absent on an intracellular sporozoite (arrowhead). The latter resides inside a PV which is visible by the surrounding halo. (D) Electrophoretic separation of the antigen precipitated by MAb 11D5/H3 from sporocyst-sporozoite extracts in gel-loading buffer containing 4% 2-mercaptoethanol (lane 2). A major band at 58 kDa was detected, whereby two minor bands appeared at 27 and 31 kDa. MAb 11D5/H3 did not react with bradyzoite extracts (lane 1), indicating that the antigen was not expressed in bradyzoites. An irrelevant isotype-matched MAb (IgG2a) and a sporozoite-specific rabbit serum (K3) served as negative and positive controls, respectively. (E) Electrophoretic separation of the antigen precipitated by 11D5/H3 from sporocyst-sporozoite extracts as performed before, except that reducing conditions were lowered to 2% 2-mercaptoethanol.

    Techniques Used: Immunofluorescence, Immunoprecipitation, Staining, Labeling

    22) Product Images from "Association between DNA Methylation in the miR-328 5'-Flanking Region and Inter-individual Differences in miR-328 and BCRP Expression in Human Placenta"

    Article Title: Association between DNA Methylation in the miR-328 5'-Flanking Region and Inter-individual Differences in miR-328 and BCRP Expression in Human Placenta

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072906

    C/EBPα-binding frequency in the miR-328 5’-flanking region. (A) Schematic representation of predicted binding sites of C/EBPα in the miR-328 5’ flanking region. The position numbers indicate the 5’ end of the C/EBPα motif. Primers for the ChIP assay are also indicated. (B) Comparison of C/EBPα-binding frequency in the miR-328 5’ flanking region. Open and closed squares indicate the results of 5-aza-dC and DMSO (control) treatment, respectively, in BeWo cells. Immunoprecipitated DNA was amplified with ten specific primers (I-X described as double-headed lines). (C) C/EBPα-binding frequency in human placentas (n = 20). Input, DNA isolated from the lysate before immunoprecipitation; IP, DNA immunoprecipitated with anti-C/EBPα antibody. Data are expressed as the mean ± SD for three independent experiments.
    Figure Legend Snippet: C/EBPα-binding frequency in the miR-328 5’-flanking region. (A) Schematic representation of predicted binding sites of C/EBPα in the miR-328 5’ flanking region. The position numbers indicate the 5’ end of the C/EBPα motif. Primers for the ChIP assay are also indicated. (B) Comparison of C/EBPα-binding frequency in the miR-328 5’ flanking region. Open and closed squares indicate the results of 5-aza-dC and DMSO (control) treatment, respectively, in BeWo cells. Immunoprecipitated DNA was amplified with ten specific primers (I-X described as double-headed lines). (C) C/EBPα-binding frequency in human placentas (n = 20). Input, DNA isolated from the lysate before immunoprecipitation; IP, DNA immunoprecipitated with anti-C/EBPα antibody. Data are expressed as the mean ± SD for three independent experiments.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Isolation

    23) Product Images from "Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation"

    Article Title: Adhesion Proteins - An Impact on Skeletal Myoblast Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061760

    Interactions beetween adhesion proteins during myoblast differentiation in vitro . A - colocalization of integrin alpha3 (green) and integrin beta1, ADAM12, CD9, CD81, M-cadherin, or VCAM-1 (red). Scale bars 50 µm. Arrows show the area where colocalization was not observed. B - Immunoprecipitation of integrin alpha3 and subsequent Western blotting analysis of CD9, CD81, M-cadherin, and V-CAM-1, c – control immunoblotting with whole cell lysate. C - interactions of analysed adhesion proteis during myoblasts differentiation. The table sumarize current study and previous results [38] .
    Figure Legend Snippet: Interactions beetween adhesion proteins during myoblast differentiation in vitro . A - colocalization of integrin alpha3 (green) and integrin beta1, ADAM12, CD9, CD81, M-cadherin, or VCAM-1 (red). Scale bars 50 µm. Arrows show the area where colocalization was not observed. B - Immunoprecipitation of integrin alpha3 and subsequent Western blotting analysis of CD9, CD81, M-cadherin, and V-CAM-1, c – control immunoblotting with whole cell lysate. C - interactions of analysed adhesion proteis during myoblasts differentiation. The table sumarize current study and previous results [38] .

    Techniques Used: In Vitro, Immunoprecipitation, Western Blot, Chick Chorioallantoic Membrane Assay

    24) Product Images from "CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath"

    Article Title: CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-8-101

    Immunoprecipitation of CABYR protein isoforms by anti-AKAP3 and anti-AKAP4 . A, B: 2-D Western blots of immunoprecipitates from mouse sperm extracts using either anti-AKAP3 polyclonal antibody (A) or pre-immune serum (B) probed with anti-CABYR-A polyclonal antibody. CABYR-A protein isoform at 80 kDa, pI 4.5 is evident in immune complexes brought down by anti-AKAP3 but not by pre-immune serum. C, D: 2-D Western blot of immunoprecipitate from mouse protein extract using anti-AKAP4 monoclonal antibody (C) or normal mouse IgG (D) probed by anti-CABYR-A polyclonal antibody. The CABYR-A only variants of 80 kDa, pI 4.5 indicated by an arrow (C) were precipitated by anti-AKAP 4 antibody while normal mouse IgG immunoprecipitated no anti-CABYR immunoreactive proteins (D). 2-D Western blots of mouse sperm extract were immunoprecipitated using anti-AKAP3 polyclonal antibody (E), anti-AKAP4 monoclonal antibody (G), pre-immune serum (F), or normal mouse IgG (H) and probed by anti-CABYR-B polyclonal antibody. The 50 kDa, pI 6.5 spots indicated by arrows in E and G represent immunoprecipitated CABYR forms that contain both CABYR-A and CABYR-B and were recognized by antibody directed at CABYR-B.
    Figure Legend Snippet: Immunoprecipitation of CABYR protein isoforms by anti-AKAP3 and anti-AKAP4 . A, B: 2-D Western blots of immunoprecipitates from mouse sperm extracts using either anti-AKAP3 polyclonal antibody (A) or pre-immune serum (B) probed with anti-CABYR-A polyclonal antibody. CABYR-A protein isoform at 80 kDa, pI 4.5 is evident in immune complexes brought down by anti-AKAP3 but not by pre-immune serum. C, D: 2-D Western blot of immunoprecipitate from mouse protein extract using anti-AKAP4 monoclonal antibody (C) or normal mouse IgG (D) probed by anti-CABYR-A polyclonal antibody. The CABYR-A only variants of 80 kDa, pI 4.5 indicated by an arrow (C) were precipitated by anti-AKAP 4 antibody while normal mouse IgG immunoprecipitated no anti-CABYR immunoreactive proteins (D). 2-D Western blots of mouse sperm extract were immunoprecipitated using anti-AKAP3 polyclonal antibody (E), anti-AKAP4 monoclonal antibody (G), pre-immune serum (F), or normal mouse IgG (H) and probed by anti-CABYR-B polyclonal antibody. The 50 kDa, pI 6.5 spots indicated by arrows in E and G represent immunoprecipitated CABYR forms that contain both CABYR-A and CABYR-B and were recognized by antibody directed at CABYR-B.

    Techniques Used: Immunoprecipitation, Western Blot

    25) Product Images from "Human Metapneumovirus M2-2 Protein Inhibits Innate Immune Response in Monocyte-Derived Dendritic Cells"

    Article Title: Human Metapneumovirus M2-2 Protein Inhibits Innate Immune Response in Monocyte-Derived Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0091865

    M2-2 interacts with MyD88. ( A–B ) M2-2 forms a complex with MyD88 in the overexpression system. 293 cells were transfected with plasmids encoding HA-tagged MyD88 and V5-tagged M2-2 or their control vectors as indicated. Total cell lysates were immunoprecipitated with an anti-V5 antibody followed by Western blotting using an anti-Flag antibody to detect MyD88 ( A ). Reverse immunoprecipitation was also done, where MyD88 was immunoprecipitated using an anti-HA antibody and M2-2 protein was then detected using an anti-V5 antibody ( B ). Membranes were stripped and reprobed to check for proper IP of M2-2 and MyD88. A small aliquot was also prepared before the IP for a Western blot for equal input of MyD88 and proper expression of M2-2 in 293 cells. ( C ) Viral M2-2 binds to endogenous MyD88 in the context of hMPV infection. THP1 cells were mock infected or infected with rhMPV-WT or -ΔM2-2, at an MOI of 5, and harvested at 24 h p.i. to prepare total cell lysates. Samples were subjected to immunoprecipitation using an anti-MyD88 antibody or control isotype. The immunoprecipitated complexes were then subjected to SDS-PAGE followed by Western blotting using an anti-hMPV antibody. The membrane was then stripped and reprobed with an anti-MyD88 antibody to determine levels of immunoprecipitated MyD88. Data are representative of two independent experiments.
    Figure Legend Snippet: M2-2 interacts with MyD88. ( A–B ) M2-2 forms a complex with MyD88 in the overexpression system. 293 cells were transfected with plasmids encoding HA-tagged MyD88 and V5-tagged M2-2 or their control vectors as indicated. Total cell lysates were immunoprecipitated with an anti-V5 antibody followed by Western blotting using an anti-Flag antibody to detect MyD88 ( A ). Reverse immunoprecipitation was also done, where MyD88 was immunoprecipitated using an anti-HA antibody and M2-2 protein was then detected using an anti-V5 antibody ( B ). Membranes were stripped and reprobed to check for proper IP of M2-2 and MyD88. A small aliquot was also prepared before the IP for a Western blot for equal input of MyD88 and proper expression of M2-2 in 293 cells. ( C ) Viral M2-2 binds to endogenous MyD88 in the context of hMPV infection. THP1 cells were mock infected or infected with rhMPV-WT or -ΔM2-2, at an MOI of 5, and harvested at 24 h p.i. to prepare total cell lysates. Samples were subjected to immunoprecipitation using an anti-MyD88 antibody or control isotype. The immunoprecipitated complexes were then subjected to SDS-PAGE followed by Western blotting using an anti-hMPV antibody. The membrane was then stripped and reprobed with an anti-MyD88 antibody to determine levels of immunoprecipitated MyD88. Data are representative of two independent experiments.

    Techniques Used: Over Expression, Transfection, Immunoprecipitation, Western Blot, Expressing, Infection, SDS Page

    26) Product Images from "Histone H1 Poly[ADP]-Ribosylation Regulates the Chromatin Alterations Required for Learning Consolidation"

    Article Title: Histone H1 Poly[ADP]-Ribosylation Regulates the Chromatin Alterations Required for Learning Consolidation

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3010-10.2010

    OR training sessions induced histone H1 poly[ADP]-ribosylation and protein decrease in the hippocampus. A , Protein extracts from the hippocampus obtained from untrained mice and those that had just finished their training, in the presence or absence of Tiq-A (0.5 mg/kg), were analyzed by two-dimensional electrophoresis, and histone H1 immunoreactivity was determined. Poly[ADP]-ribosylation of histone H1 is reflected by a shift in pI toward acidic pH values. B , Hippocampal protein extracts from untrained and trained mice that received Tiq-A (0.5 mg/kg) or the vehicle alone and were killed 15 min after training session were used to immunoprecipitate PAR polymer or histone H1. The immunoprecipitates were examined in Western blots probed for histone H1 and PAR polymer (bottom). As an internal control, 10% of the protein used for the immunoprecipitation assays was examined in Western blots probed for PAR polymer, histone H1, and actin (top). Values illustrate the densitometric quantification of PAR polymer and histone H1 immunoreactivity in each experimental group or the relative amount of histone H1 poly[ADP]-ribosylated (PAR-histone H1) in each experimental group with respect to the untrained animals. C , Immunohistochemistry of histone H1 expression in the hippocampal CA1 field of mice killed 1 h after training or manipulation in the presence or absence of Tiq-A. The top shows representative images of histone H1 in CA1 neurons after the manipulations. The bottom shows a bar graph of the percentage of neurons labeled with high or low amounts of histone H1 under the different conditions ( n = 5 mice per groups). IP, Immunoprecipitation. ** p
    Figure Legend Snippet: OR training sessions induced histone H1 poly[ADP]-ribosylation and protein decrease in the hippocampus. A , Protein extracts from the hippocampus obtained from untrained mice and those that had just finished their training, in the presence or absence of Tiq-A (0.5 mg/kg), were analyzed by two-dimensional electrophoresis, and histone H1 immunoreactivity was determined. Poly[ADP]-ribosylation of histone H1 is reflected by a shift in pI toward acidic pH values. B , Hippocampal protein extracts from untrained and trained mice that received Tiq-A (0.5 mg/kg) or the vehicle alone and were killed 15 min after training session were used to immunoprecipitate PAR polymer or histone H1. The immunoprecipitates were examined in Western blots probed for histone H1 and PAR polymer (bottom). As an internal control, 10% of the protein used for the immunoprecipitation assays was examined in Western blots probed for PAR polymer, histone H1, and actin (top). Values illustrate the densitometric quantification of PAR polymer and histone H1 immunoreactivity in each experimental group or the relative amount of histone H1 poly[ADP]-ribosylated (PAR-histone H1) in each experimental group with respect to the untrained animals. C , Immunohistochemistry of histone H1 expression in the hippocampal CA1 field of mice killed 1 h after training or manipulation in the presence or absence of Tiq-A. The top shows representative images of histone H1 in CA1 neurons after the manipulations. The bottom shows a bar graph of the percentage of neurons labeled with high or low amounts of histone H1 under the different conditions ( n = 5 mice per groups). IP, Immunoprecipitation. ** p

    Techniques Used: Mouse Assay, Electrophoresis, Western Blot, Immunoprecipitation, Immunohistochemistry, Expressing, Labeling

    OR training promotes PARP-1-dependent histone H1 clearance and RNA pol II recruitment to NF-κB and CREB gene promoters. A , Semiquantitative reverse transcription-PCR analysis of i-nos, tnf-α, c-fos, c-jun, and egr-1 mRNA from the hippocampus of untrained mice and from that obtained 1 h after OR training in mice that received Tiq-A or the vehicle alone. GAPDH mRNA served as an internal control. The bar graph represents the trained/untrained ratio of the normalized values of gene expression in the presence or absence of Tiq-A (white and black bars, respectively; n = 5 mice per group). B , C , The levels of histone H1 ( B ) and RNA pol II ( C ) at the tnf-α, c-fos, c-jun, and egr-1 promoters were studied by chromatin immunoprecipitation in hippocampal chromatin complexes obtained from untrained and trained mice that received Tiq-A or the vehicle alone. Histograms represent the trained/untrained ratio of normalized values of gene promoter amplification in the presence or absence of Tiq-A (black and white bars, respectively; n = 3 mice per group). Asterisks above the line indicate a difference between the two treatments (vehicle and Tiq-A), whereas asterisks above a bar graph indicate a significant difference between trained and untrained mice. * p
    Figure Legend Snippet: OR training promotes PARP-1-dependent histone H1 clearance and RNA pol II recruitment to NF-κB and CREB gene promoters. A , Semiquantitative reverse transcription-PCR analysis of i-nos, tnf-α, c-fos, c-jun, and egr-1 mRNA from the hippocampus of untrained mice and from that obtained 1 h after OR training in mice that received Tiq-A or the vehicle alone. GAPDH mRNA served as an internal control. The bar graph represents the trained/untrained ratio of the normalized values of gene expression in the presence or absence of Tiq-A (white and black bars, respectively; n = 5 mice per group). B , C , The levels of histone H1 ( B ) and RNA pol II ( C ) at the tnf-α, c-fos, c-jun, and egr-1 promoters were studied by chromatin immunoprecipitation in hippocampal chromatin complexes obtained from untrained and trained mice that received Tiq-A or the vehicle alone. Histograms represent the trained/untrained ratio of normalized values of gene promoter amplification in the presence or absence of Tiq-A (black and white bars, respectively; n = 3 mice per group). Asterisks above the line indicate a difference between the two treatments (vehicle and Tiq-A), whereas asterisks above a bar graph indicate a significant difference between trained and untrained mice. * p

    Techniques Used: Polymerase Chain Reaction, Mouse Assay, Expressing, Chromatin Immunoprecipitation, Amplification

    27) Product Images from "Inhibition of Cyclin-Dependent Kinase Phosphorylation of FOXO1 and Prostate Cancer Cell Growth by a Peptide Derived from FOXO1 1"

    Article Title: Inhibition of Cyclin-Dependent Kinase Phosphorylation of FOXO1 and Prostate Cancer Cell Growth by a Peptide Derived from FOXO1 1

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

    doi:

    FO1-6nls inhibits phosphorylation of FOXO1 at S249 in PCa cells. (A) DU145 cells grown in the exponential phase were harvested and lysed for immunoprecipitation with anti-FOXO1 antibodies and Western blot analysis with antibodies as indicated. Immunoblot
    Figure Legend Snippet: FO1-6nls inhibits phosphorylation of FOXO1 at S249 in PCa cells. (A) DU145 cells grown in the exponential phase were harvested and lysed for immunoprecipitation with anti-FOXO1 antibodies and Western blot analysis with antibodies as indicated. Immunoblot

    Techniques Used: Immunoprecipitation, Western Blot

    28) Product Images from "Suppressor of Cytokine Signaling 1 Expression Protects Oligodendrocytes from the Deleterious Effects of Interferon-γ"

    Article Title: Suppressor of Cytokine Signaling 1 Expression Protects Oligodendrocytes from the Deleterious Effects of Interferon-γ

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0737-06.2006

    Expression of Flag-SOCS1. A , PLP/SOCS1 construct contains 2.4 kb of the PLP 5′ flanking DNA, exon 1 (no ATG), intron 1 (diagonally striped boxes), Flag-SOCS1, and simian virus 40 (SV40) poly(A) signal sequence. Expression of the PLP/SOCS1 transgene was characterized at postnatal day 21 using several methods. B , Northern blot analysis demonstrated Flag-SOCS1 expression in PLP/SOCS1 brain (lane 2; T, transgenic brain) compared with wild-type brain (lane 1; W, wild-type brain). C , Q-PCR analysis with transgene-specific primers revealed the highest concentrations of transgene-derived SOCS1 mRNA were in the brain, spinal cord, and sciatic nerve, with significantly lower levels in other organs. D , Western blot. E , Immunoprecipitation. Both demonstrated a single 19 kDa Flag-positive band, the expected molecular weight (MW) of SOCS1, only in the lanes loaded with brain samples from PLP/SOCS1 mice (brain fSOCS1+). Flag protein was used as a positive control for the antibody reaction; 15% SDS-PAGE, anti-Flag (M2) antibody. Immunostaining with anti-SOCS1/FITC ( F , H , green) and anti-Flag/FITC ( G , I , green) antibodies demonstrated positive signal only in PLP/SOCS1 ( H , I , green) and not in the wild-type mouse samples ( F , G ). Cell nuclei were contrastained with ethidium bromide ( F–I , red). Coronal sections of thalamic fiber are shown. Scale bar, 20 μm.
    Figure Legend Snippet: Expression of Flag-SOCS1. A , PLP/SOCS1 construct contains 2.4 kb of the PLP 5′ flanking DNA, exon 1 (no ATG), intron 1 (diagonally striped boxes), Flag-SOCS1, and simian virus 40 (SV40) poly(A) signal sequence. Expression of the PLP/SOCS1 transgene was characterized at postnatal day 21 using several methods. B , Northern blot analysis demonstrated Flag-SOCS1 expression in PLP/SOCS1 brain (lane 2; T, transgenic brain) compared with wild-type brain (lane 1; W, wild-type brain). C , Q-PCR analysis with transgene-specific primers revealed the highest concentrations of transgene-derived SOCS1 mRNA were in the brain, spinal cord, and sciatic nerve, with significantly lower levels in other organs. D , Western blot. E , Immunoprecipitation. Both demonstrated a single 19 kDa Flag-positive band, the expected molecular weight (MW) of SOCS1, only in the lanes loaded with brain samples from PLP/SOCS1 mice (brain fSOCS1+). Flag protein was used as a positive control for the antibody reaction; 15% SDS-PAGE, anti-Flag (M2) antibody. Immunostaining with anti-SOCS1/FITC ( F , H , green) and anti-Flag/FITC ( G , I , green) antibodies demonstrated positive signal only in PLP/SOCS1 ( H , I , green) and not in the wild-type mouse samples ( F , G ). Cell nuclei were contrastained with ethidium bromide ( F–I , red). Coronal sections of thalamic fiber are shown. Scale bar, 20 μm.

    Techniques Used: Expressing, Plasmid Purification, Construct, Sequencing, Northern Blot, Transgenic Assay, Polymerase Chain Reaction, Derivative Assay, Western Blot, Immunoprecipitation, Molecular Weight, Mouse Assay, Positive Control, SDS Page, Immunostaining

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    Article Snippet: Chromatin immunoprecipitation (ChIP) assay ChIP analysis was performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). .. Briefly, EPCs after 24 h treatment with 10 ng/mL IL-1β or control vehicle (DMSO) were fixed by adding formaldehyde to a final concentration of 1% at 37°C for 10 min and then washed with ice-cold PBS twice, collected by centrifugation (5 min at 2000 × g ) and resuspended in 1 mL of SDS lysis buffer containing 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). .. Cell lysate was sonicated (5 times with 10-s pulses and 1-min breaks) on ice with a 60 Sonic Dismembrator (Fisher Scientific), followed by centrifugation at 4°C for 10 min. Immunoprecipitations were carried out according to the manufacturer’s protocol.

    Centrifugation:

    Article Title: C/EBP? Mediates Osteoclast Recruitment by Regulating Endothelial Progenitor Cell Expression of SDF-1?
    Article Snippet: Chromatin immunoprecipitation (ChIP) assay ChIP analysis was performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). .. Briefly, EPCs after 24 h treatment with 10 ng/mL IL-1β or control vehicle (DMSO) were fixed by adding formaldehyde to a final concentration of 1% at 37°C for 10 min and then washed with ice-cold PBS twice, collected by centrifugation (5 min at 2000 × g ) and resuspended in 1 mL of SDS lysis buffer containing 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). .. Cell lysate was sonicated (5 times with 10-s pulses and 1-min breaks) on ice with a 60 Sonic Dismembrator (Fisher Scientific), followed by centrifugation at 4°C for 10 min. Immunoprecipitations were carried out according to the manufacturer’s protocol.

    Lysis:

    Article Title: C/EBP? Mediates Osteoclast Recruitment by Regulating Endothelial Progenitor Cell Expression of SDF-1?
    Article Snippet: Chromatin immunoprecipitation (ChIP) assay ChIP analysis was performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). .. Briefly, EPCs after 24 h treatment with 10 ng/mL IL-1β or control vehicle (DMSO) were fixed by adding formaldehyde to a final concentration of 1% at 37°C for 10 min and then washed with ice-cold PBS twice, collected by centrifugation (5 min at 2000 × g ) and resuspended in 1 mL of SDS lysis buffer containing 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). .. Cell lysate was sonicated (5 times with 10-s pulses and 1-min breaks) on ice with a 60 Sonic Dismembrator (Fisher Scientific), followed by centrifugation at 4°C for 10 min. Immunoprecipitations were carried out according to the manufacturer’s protocol.

    Article Title: Novel AKT1-GLI3-VMP1 Pathway Mediates KRAS Oncogene-induced Autophagy in Cancer Cells *
    Article Snippet: Punctate fluorescence was imaged by fluorescence microscopy using an LSM510 microscope (Zeiss, Heidelberg, Germany). .. PANC1 cells were washed twice with cold phosphate-buffered saline and lysed in lysis buffer (150 m m NaCl, 0.5% Nonidet P-40, 50 m m Tris-HCl, pH 7.5, 20 m m MgCl2 ) supplemented with Complete protease inhibitor tablets (Roche Applied Science, Penzberg, Germany) for 1 h on ice. .. After the lysates were cleared at 15,000 × g for 20 min, supernatants were collected and subjected to immunoprecipitation following the Dynabeads Protein G immunoprecipitation kit protocol (Invitrogen).

    Protease Inhibitor:

    Article Title: C/EBP? Mediates Osteoclast Recruitment by Regulating Endothelial Progenitor Cell Expression of SDF-1?
    Article Snippet: Chromatin immunoprecipitation (ChIP) assay ChIP analysis was performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). .. Briefly, EPCs after 24 h treatment with 10 ng/mL IL-1β or control vehicle (DMSO) were fixed by adding formaldehyde to a final concentration of 1% at 37°C for 10 min and then washed with ice-cold PBS twice, collected by centrifugation (5 min at 2000 × g ) and resuspended in 1 mL of SDS lysis buffer containing 1 × protease inhibitor cocktail (Roche, Rotkreuz, Switzerland). .. Cell lysate was sonicated (5 times with 10-s pulses and 1-min breaks) on ice with a 60 Sonic Dismembrator (Fisher Scientific), followed by centrifugation at 4°C for 10 min. Immunoprecipitations were carried out according to the manufacturer’s protocol.

    Article Title: Novel AKT1-GLI3-VMP1 Pathway Mediates KRAS Oncogene-induced Autophagy in Cancer Cells *
    Article Snippet: Punctate fluorescence was imaged by fluorescence microscopy using an LSM510 microscope (Zeiss, Heidelberg, Germany). .. PANC1 cells were washed twice with cold phosphate-buffered saline and lysed in lysis buffer (150 m m NaCl, 0.5% Nonidet P-40, 50 m m Tris-HCl, pH 7.5, 20 m m MgCl2 ) supplemented with Complete protease inhibitor tablets (Roche Applied Science, Penzberg, Germany) for 1 h on ice. .. After the lysates were cleared at 15,000 × g for 20 min, supernatants were collected and subjected to immunoprecipitation following the Dynabeads Protein G immunoprecipitation kit protocol (Invitrogen).

    End Labeling:

    Article Title: Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression
    Article Snippet: The recovered DNA was analyzed by PCR for the binding of STAT3, p65, and H3K4-me at the mouse Smyd2 promoter. .. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayTUNEL assays for AZ505-treated or SMYD2 knockdown cells and for AZ505–treated or shRNA-SMYD2 knockdown xenografts were performed according to the manufacturer’s protocols (In Situ Death Detection Kit; Roche). .. Prolong Gold Anti-fade reagent with DAPI (Invitrogen) was used.

    TUNEL Assay:

    Article Title: Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression
    Article Snippet: The recovered DNA was analyzed by PCR for the binding of STAT3, p65, and H3K4-me at the mouse Smyd2 promoter. .. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayTUNEL assays for AZ505-treated or SMYD2 knockdown cells and for AZ505–treated or shRNA-SMYD2 knockdown xenografts were performed according to the manufacturer’s protocols (In Situ Death Detection Kit; Roche). .. Prolong Gold Anti-fade reagent with DAPI (Invitrogen) was used.

    Article Title: Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor
    Article Snippet: The antibody of Egr-1 was used for immunoprecipitation and the primer set (5′-CAG AGG TCA CTT AAG TAC CCA GCC ATC T-3′ and 5′-ATA AGC TTT GCA GTC TGC CTG AGC ATC C-3′) were used for amplify of mouse PlGF promoter according to the manufacturer's instructions. .. TUNEL assay Treated or untreated MLE-15 cells and OCT-embedded lung tissue from the mice were analyzed for the level of apoptosis using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions, and the fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany). .. Trypan blue inclusion assay Treated or untreated MLE-15 cells were treated with 0–100 ng/ml PlGF for 24 h then stained with trypan blue (1 : 1 in volume), and the cells that stained blue were counted using a hemacytometer counting chamber (Invitrogen).

    shRNA:

    Article Title: Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression
    Article Snippet: The recovered DNA was analyzed by PCR for the binding of STAT3, p65, and H3K4-me at the mouse Smyd2 promoter. .. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayTUNEL assays for AZ505-treated or SMYD2 knockdown cells and for AZ505–treated or shRNA-SMYD2 knockdown xenografts were performed according to the manufacturer’s protocols (In Situ Death Detection Kit; Roche). .. Prolong Gold Anti-fade reagent with DAPI (Invitrogen) was used.

    In Situ:

    Article Title: Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression
    Article Snippet: The recovered DNA was analyzed by PCR for the binding of STAT3, p65, and H3K4-me at the mouse Smyd2 promoter. .. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayTUNEL assays for AZ505-treated or SMYD2 knockdown cells and for AZ505–treated or shRNA-SMYD2 knockdown xenografts were performed according to the manufacturer’s protocols (In Situ Death Detection Kit; Roche). .. Prolong Gold Anti-fade reagent with DAPI (Invitrogen) was used.

    Article Title: Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor
    Article Snippet: The antibody of Egr-1 was used for immunoprecipitation and the primer set (5′-CAG AGG TCA CTT AAG TAC CCA GCC ATC T-3′ and 5′-ATA AGC TTT GCA GTC TGC CTG AGC ATC C-3′) were used for amplify of mouse PlGF promoter according to the manufacturer's instructions. .. TUNEL assay Treated or untreated MLE-15 cells and OCT-embedded lung tissue from the mice were analyzed for the level of apoptosis using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions, and the fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany). .. Trypan blue inclusion assay Treated or untreated MLE-15 cells were treated with 0–100 ng/ml PlGF for 24 h then stained with trypan blue (1 : 1 in volume), and the cells that stained blue were counted using a hemacytometer counting chamber (Invitrogen).

    Plasmid Preparation:

    Article Title: Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus
    Article Snippet: Despite five point mutations (5′-GGA A GA G GT C AT T CG C AAA-3′, mutated nucleotides are underlined) and also according to the genetic code, the new cDNA sequence encodes a functional Mbd2 protein. .. This vector was generated by digesting the pCMV-MBD2 vector (kindly provided by Dr A. Bird) within the Mbd2 cDNA sequence with KspI (Roche) and AccIII (Promega) endonucleases. .. This KspI/AccIII fragment was gel-purified (MinElute gel purification Kit; Qiagen) and digested by ApaI and AccI (Roche).

    Generated:

    Article Title: Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus
    Article Snippet: Despite five point mutations (5′-GGA A GA G GT C AT T CG C AAA-3′, mutated nucleotides are underlined) and also according to the genetic code, the new cDNA sequence encodes a functional Mbd2 protein. .. This vector was generated by digesting the pCMV-MBD2 vector (kindly provided by Dr A. Bird) within the Mbd2 cDNA sequence with KspI (Roche) and AccIII (Promega) endonucleases. .. This KspI/AccIII fragment was gel-purified (MinElute gel purification Kit; Qiagen) and digested by ApaI and AccI (Roche).

    Sequencing:

    Article Title: Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus
    Article Snippet: Despite five point mutations (5′-GGA A GA G GT C AT T CG C AAA-3′, mutated nucleotides are underlined) and also according to the genetic code, the new cDNA sequence encodes a functional Mbd2 protein. .. This vector was generated by digesting the pCMV-MBD2 vector (kindly provided by Dr A. Bird) within the Mbd2 cDNA sequence with KspI (Roche) and AccIII (Promega) endonucleases. .. This KspI/AccIII fragment was gel-purified (MinElute gel purification Kit; Qiagen) and digested by ApaI and AccI (Roche).

    Article Title: Local and global chromatin interactions are altered by large genomic deletions associated with human brain development
    Article Snippet: The DNA fragments were then amplified and purified. .. A total of 2.1 million oligomer capture probes that densely tile the sequence of chromosome 22q were designed using the NimbleDesign Software and ordered from Roche Sequencing (formerly NimbleGen-Roche, Pleasanton, CA). .. Roche Sequencing’s SeqCap EZ Choice XL Enrichment Kit was used to capture fragments from the in situ Hi-C libraries using the chromosome 22q-specific capture oligomers.

    Mouse Assay:

    Article Title: Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor
    Article Snippet: The antibody of Egr-1 was used for immunoprecipitation and the primer set (5′-CAG AGG TCA CTT AAG TAC CCA GCC ATC T-3′ and 5′-ATA AGC TTT GCA GTC TGC CTG AGC ATC C-3′) were used for amplify of mouse PlGF promoter according to the manufacturer's instructions. .. TUNEL assay Treated or untreated MLE-15 cells and OCT-embedded lung tissue from the mice were analyzed for the level of apoptosis using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions, and the fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany). .. Trypan blue inclusion assay Treated or untreated MLE-15 cells were treated with 0–100 ng/ml PlGF for 24 h then stained with trypan blue (1 : 1 in volume), and the cells that stained blue were counted using a hemacytometer counting chamber (Invitrogen).

    Fluorescence:

    Article Title: Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor
    Article Snippet: The antibody of Egr-1 was used for immunoprecipitation and the primer set (5′-CAG AGG TCA CTT AAG TAC CCA GCC ATC T-3′ and 5′-ATA AGC TTT GCA GTC TGC CTG AGC ATC C-3′) were used for amplify of mouse PlGF promoter according to the manufacturer's instructions. .. TUNEL assay Treated or untreated MLE-15 cells and OCT-embedded lung tissue from the mice were analyzed for the level of apoptosis using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions, and the fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany). .. Trypan blue inclusion assay Treated or untreated MLE-15 cells were treated with 0–100 ng/ml PlGF for 24 h then stained with trypan blue (1 : 1 in volume), and the cells that stained blue were counted using a hemacytometer counting chamber (Invitrogen).

    Microscopy:

    Article Title: Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor
    Article Snippet: The antibody of Egr-1 was used for immunoprecipitation and the primer set (5′-CAG AGG TCA CTT AAG TAC CCA GCC ATC T-3′ and 5′-ATA AGC TTT GCA GTC TGC CTG AGC ATC C-3′) were used for amplify of mouse PlGF promoter according to the manufacturer's instructions. .. TUNEL assay Treated or untreated MLE-15 cells and OCT-embedded lung tissue from the mice were analyzed for the level of apoptosis using an In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions, and the fluorescence-positive cells were photographed by a Leica DM 4000B microscope (Leica, Solms, Germany). .. Trypan blue inclusion assay Treated or untreated MLE-15 cells were treated with 0–100 ng/ml PlGF for 24 h then stained with trypan blue (1 : 1 in volume), and the cells that stained blue were counted using a hemacytometer counting chamber (Invitrogen).

    Construct:

    Article Title: Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane
    Article Snippet: Mutants were created by QuikChange kit (Stratagene, La Jolla, CA). .. Constructs were stably transfected into CHO-K1 cells using FuGENE transfection kit ((Roche Diagnostics, Indianapolis, IN). .. The expression levels of αIIb β3 were determined by flow cytometry staining with the following monoclonal antibodies (mAbs): AP3 (anti-β3 mAb, American Type Culture Collection), 7E3 (anti-β3 mAb), and 10E5 (anti-αIIb mAb, gifted by B. S. Coller, Rockefeller University, NY).

    Stable Transfection:

    Article Title: Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane
    Article Snippet: Mutants were created by QuikChange kit (Stratagene, La Jolla, CA). .. Constructs were stably transfected into CHO-K1 cells using FuGENE transfection kit ((Roche Diagnostics, Indianapolis, IN). .. The expression levels of αIIb β3 were determined by flow cytometry staining with the following monoclonal antibodies (mAbs): AP3 (anti-β3 mAb, American Type Culture Collection), 7E3 (anti-β3 mAb), and 10E5 (anti-αIIb mAb, gifted by B. S. Coller, Rockefeller University, NY).

    Transfection:

    Article Title: Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane
    Article Snippet: Mutants were created by QuikChange kit (Stratagene, La Jolla, CA). .. Constructs were stably transfected into CHO-K1 cells using FuGENE transfection kit ((Roche Diagnostics, Indianapolis, IN). .. The expression levels of αIIb β3 were determined by flow cytometry staining with the following monoclonal antibodies (mAbs): AP3 (anti-β3 mAb, American Type Culture Collection), 7E3 (anti-β3 mAb), and 10E5 (anti-αIIb mAb, gifted by B. S. Coller, Rockefeller University, NY).

    Software:

    Article Title: Local and global chromatin interactions are altered by large genomic deletions associated with human brain development
    Article Snippet: The DNA fragments were then amplified and purified. .. A total of 2.1 million oligomer capture probes that densely tile the sequence of chromosome 22q were designed using the NimbleDesign Software and ordered from Roche Sequencing (formerly NimbleGen-Roche, Pleasanton, CA). .. Roche Sequencing’s SeqCap EZ Choice XL Enrichment Kit was used to capture fragments from the in situ Hi-C libraries using the chromosome 22q-specific capture oligomers.

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    Roche lightcycler 480 sybr green i master mix kit
    Lack of the H4 C-terminal tail but not H4 G94P alters anti-silencing function 1 (Asf1)-mediated disome formation. H3/H4 G94P (A) or H3/H4 ∆94 (B) were compared to wild-type (WT) H3/H4, all at 0.8 μM dimer concentration, for their ability to form tetrasomes and disomes on 80 bp 5 S DNA (0.4 μM) in the absence and presence of Asf1 (0, 0.8, 2.0 μM). Upper panels show images of <t>SYBR</t> <t>Green</t> I stained DNA, and lower panels show the quantitation of the amount of disomes and tetrasomes formed for each type of histone, from at least three independent experiments. Tetrasome and disome levels were normalized to the WT H3/H4 sample in the absence of Asf1.
    Lightcycler 480 Sybr Green I Master Mix Kit, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Roche immunoprecipitation kit
    Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by <t>co-immunoprecipitation</t> using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.
    Immunoprecipitation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoprecipitation kit/product/Roche
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    86
    Roche high pure ffpe rna micro kit
    Development of a high-throughput microfluidic gene expression assay for analysis of <t>FFPE</t> breast tumor samples. (A) Schematic of the gene expression assay protocol. (B) Representative five-point standard curves of FFPE tumor <t>RNA</t> (red and black lines) and universal RNA (blue line) run on the breast cancer gene expression assay (slope of line indicated). (C) Intra-chip reproducibility of FFPE tumor samples run on the same 96.96 Dynamic Array. (D) Inter-chip reproducibility of the breast cancer gene expression assay assessed by comparing Ct values of universal RNA across seven independent assay runs. R-squared values are indicated in boxes.
    High Pure Ffpe Rna Micro Kit, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche lightcycler 480 sybr green i master kit mix
    Binding determinations of NRF-1, KLF15 and Sp1 transcription factors to the hPCFT minimal promoter region by ChIP and EMSA assays. ( A ) ChIP assays were performed in HepG2 and HT1080 cells according to the manufacturer’s instructions, with antibodies against NRF-1, KLF15, and Sp1. Mouse IgG2a was used as a negative control in the pull-down step. The ChIP DNA and input DNA were prepared with the ChIP-IT High Sensitivity Kit according to the manufacturer’s instructions and were then used for real-time PCR analyses with the ‘DNA Standards, Design and Analysis Template’ provided with the ChIP-IT qPCR Analysis kit (Active Motif). Real-time PCR was performed using a Roche <t>LightCycler</t> 480 and LightCycler 480 <t>SYBR</t> <t>Green</t> I Master kit, with primers located in the hPCFT core promoter region spanning putative binding sites for NRF-1, KLF15 or Sp1. Statistical analyses were performed using Prism 6.07. Results are presented as mean values ± standard errors from at least three independent experiments of triplicate measurements. **** p
    Lightcycler 480 Sybr Green I Master Kit Mix, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lightcycler 480 sybr green i master kit mix/product/Roche
    Average 86 stars, based on 1 article reviews
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    Image Search Results


    Lack of the H4 C-terminal tail but not H4 G94P alters anti-silencing function 1 (Asf1)-mediated disome formation. H3/H4 G94P (A) or H3/H4 ∆94 (B) were compared to wild-type (WT) H3/H4, all at 0.8 μM dimer concentration, for their ability to form tetrasomes and disomes on 80 bp 5 S DNA (0.4 μM) in the absence and presence of Asf1 (0, 0.8, 2.0 μM). Upper panels show images of SYBR Green I stained DNA, and lower panels show the quantitation of the amount of disomes and tetrasomes formed for each type of histone, from at least three independent experiments. Tetrasome and disome levels were normalized to the WT H3/H4 sample in the absence of Asf1.

    Journal: Epigenetics & Chromatin

    Article Title: The conformational flexibility of the C-terminus of histone H4 promotes histone octamer and nucleosome stability and yeast viability

    doi: 10.1186/1756-8935-5-5

    Figure Lengend Snippet: Lack of the H4 C-terminal tail but not H4 G94P alters anti-silencing function 1 (Asf1)-mediated disome formation. H3/H4 G94P (A) or H3/H4 ∆94 (B) were compared to wild-type (WT) H3/H4, all at 0.8 μM dimer concentration, for their ability to form tetrasomes and disomes on 80 bp 5 S DNA (0.4 μM) in the absence and presence of Asf1 (0, 0.8, 2.0 μM). Upper panels show images of SYBR Green I stained DNA, and lower panels show the quantitation of the amount of disomes and tetrasomes formed for each type of histone, from at least three independent experiments. Tetrasome and disome levels were normalized to the WT H3/H4 sample in the absence of Asf1.

    Article Snippet: ChIP quantification was performed via real-time PCR with a Roche Applied Sciences LightCycler 480 and the LightCycler 480 SYBR Green I Master Mix kit (Roche catalog no. 04707516001).

    Techniques: Concentration Assay, SYBR Green Assay, Staining, Quantitation Assay

    Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by co-immunoprecipitation using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.

    Journal: Cell death and differentiation

    Article Title: Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis

    doi: 10.1038/cdd.2009.86

    Figure Lengend Snippet: Cyclin D1 interacts with FOXO1 in vitro and in vivo . ( a ) Interaction of endogenous cyclin D1 and FOXO1 proteins in LNCaP cells as shown by co-immunoprecipitation using an antibody against cyclin D1. ( b ) LNCaP cells were transfected with plasmids as indicated.

    Article Snippet: Protein immunoprecipitations were carried out using an immunoprecipitation kit (Roche Applied Sciences, Indianapolis, IN, USA) as described.

    Techniques: In Vitro, In Vivo, Immunoprecipitation, Transfection

    Development of a high-throughput microfluidic gene expression assay for analysis of FFPE breast tumor samples. (A) Schematic of the gene expression assay protocol. (B) Representative five-point standard curves of FFPE tumor RNA (red and black lines) and universal RNA (blue line) run on the breast cancer gene expression assay (slope of line indicated). (C) Intra-chip reproducibility of FFPE tumor samples run on the same 96.96 Dynamic Array. (D) Inter-chip reproducibility of the breast cancer gene expression assay assessed by comparing Ct values of universal RNA across seven independent assay runs. R-squared values are indicated in boxes.

    Journal: PLoS ONE

    Article Title: Targeted Biomarker Profiling of Matched Primary and Metastatic Estrogen Receptor Positive Breast Cancers

    doi: 10.1371/journal.pone.0088401

    Figure Lengend Snippet: Development of a high-throughput microfluidic gene expression assay for analysis of FFPE breast tumor samples. (A) Schematic of the gene expression assay protocol. (B) Representative five-point standard curves of FFPE tumor RNA (red and black lines) and universal RNA (blue line) run on the breast cancer gene expression assay (slope of line indicated). (C) Intra-chip reproducibility of FFPE tumor samples run on the same 96.96 Dynamic Array. (D) Inter-chip reproducibility of the breast cancer gene expression assay assessed by comparing Ct values of universal RNA across seven independent assay runs. R-squared values are indicated in boxes.

    Article Snippet: RNA was isolated using the High Pure FFPE RNA Micro Kit (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s protocol.

    Techniques: High Throughput Screening Assay, Expressing, Formalin-fixed Paraffin-Embedded, Chromatin Immunoprecipitation

    Binding determinations of NRF-1, KLF15 and Sp1 transcription factors to the hPCFT minimal promoter region by ChIP and EMSA assays. ( A ) ChIP assays were performed in HepG2 and HT1080 cells according to the manufacturer’s instructions, with antibodies against NRF-1, KLF15, and Sp1. Mouse IgG2a was used as a negative control in the pull-down step. The ChIP DNA and input DNA were prepared with the ChIP-IT High Sensitivity Kit according to the manufacturer’s instructions and were then used for real-time PCR analyses with the ‘DNA Standards, Design and Analysis Template’ provided with the ChIP-IT qPCR Analysis kit (Active Motif). Real-time PCR was performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master kit, with primers located in the hPCFT core promoter region spanning putative binding sites for NRF-1, KLF15 or Sp1. Statistical analyses were performed using Prism 6.07. Results are presented as mean values ± standard errors from at least three independent experiments of triplicate measurements. **** p

    Journal: The Biochemical journal

    Article Title: Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

    doi: 10.1042/BCJ20180394

    Figure Lengend Snippet: Binding determinations of NRF-1, KLF15 and Sp1 transcription factors to the hPCFT minimal promoter region by ChIP and EMSA assays. ( A ) ChIP assays were performed in HepG2 and HT1080 cells according to the manufacturer’s instructions, with antibodies against NRF-1, KLF15, and Sp1. Mouse IgG2a was used as a negative control in the pull-down step. The ChIP DNA and input DNA were prepared with the ChIP-IT High Sensitivity Kit according to the manufacturer’s instructions and were then used for real-time PCR analyses with the ‘DNA Standards, Design and Analysis Template’ provided with the ChIP-IT qPCR Analysis kit (Active Motif). Real-time PCR was performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master kit, with primers located in the hPCFT core promoter region spanning putative binding sites for NRF-1, KLF15 or Sp1. Statistical analyses were performed using Prism 6.07. Results are presented as mean values ± standard errors from at least three independent experiments of triplicate measurements. **** p

    Article Snippet: The qPCRs were performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master Kit Mix (Roche Diagnostics), with primers located in the hPCFT core promoter region that includes putative binding sites for NRF-1, KLF15 or Sp1.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, SYBR Green Assay

    NRF-1, KLF15, and Sp1 expression in HepG2 and HT1080 cells and the impact of NRF-1, KLF15 and Sp1 on hPCFT gene expression. ( A ) Transcript levels for KLF15, NRF-1 and Sp1 were measured in HepG2 and HT1080 cell lines. ( B ) Impact of hPCFT gene expression in HepG2 cells stably transfected with KLF15, NRF-1 or Sp1. ( C ) Impact of hPCFT gene expression in HT1080 cells stably transfected with KLF15, NRF-1 or Sp1. ( D ) Impact of hPCFT gene expression in HT1080 cells transiently transfected with KLF15, NRF-1 or Sp1 singly or in combination. Transcript levels of NRF-1, KLF15, Sp1 and hPCFT were monitored by real-time RT-PCR with a LightCycler 480 Probes Master kit, or with a LightCycler 480 SYBR Green I Master kit. The transcript levels were normalized to that for GAPDH and/or β-actin. Results are presented as mean values ± standard errors from three to four different experiments. For statistics: *** p

    Journal: The Biochemical journal

    Article Title: Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

    doi: 10.1042/BCJ20180394

    Figure Lengend Snippet: NRF-1, KLF15, and Sp1 expression in HepG2 and HT1080 cells and the impact of NRF-1, KLF15 and Sp1 on hPCFT gene expression. ( A ) Transcript levels for KLF15, NRF-1 and Sp1 were measured in HepG2 and HT1080 cell lines. ( B ) Impact of hPCFT gene expression in HepG2 cells stably transfected with KLF15, NRF-1 or Sp1. ( C ) Impact of hPCFT gene expression in HT1080 cells stably transfected with KLF15, NRF-1 or Sp1. ( D ) Impact of hPCFT gene expression in HT1080 cells transiently transfected with KLF15, NRF-1 or Sp1 singly or in combination. Transcript levels of NRF-1, KLF15, Sp1 and hPCFT were monitored by real-time RT-PCR with a LightCycler 480 Probes Master kit, or with a LightCycler 480 SYBR Green I Master kit. The transcript levels were normalized to that for GAPDH and/or β-actin. Results are presented as mean values ± standard errors from three to four different experiments. For statistics: *** p

    Article Snippet: The qPCRs were performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master Kit Mix (Roche Diagnostics), with primers located in the hPCFT core promoter region that includes putative binding sites for NRF-1, KLF15 or Sp1.

    Techniques: Expressing, Stable Transfection, Transfection, Quantitative RT-PCR, SYBR Green Assay

    Expression and regulatory associations between hPCFT and NRF-1, Sp1, or KLF15 in solid tumor cell lines. ) by real-time RT-PCR with LightCycler 480 SYBR Green I Master kit. The hPCFT, NRF-1, Sp1, and KLF15 transcript levels were normalized to transcript levels for GAPDH. The scatter plots show univariate associations between hPCFT and NRF-1 ( A ), hPCFT and Sp1 ( B ), and hPCFT and KLF15 ( C ) by Pearson’s correlation coefficients ( r ). The x-axis and y-axis scales are logarithmic and the solid gray lines represent the fitted regression lines. N indicates the sample size (53). ( D ]. For the schematic shown, the thickness of the edge and the corresponding numbers (which range from 0 to 1) represent the relative strength of the association. The R/Bioconductor packages minet and igraph ].

    Journal: The Biochemical journal

    Article Title: Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

    doi: 10.1042/BCJ20180394

    Figure Lengend Snippet: Expression and regulatory associations between hPCFT and NRF-1, Sp1, or KLF15 in solid tumor cell lines. ) by real-time RT-PCR with LightCycler 480 SYBR Green I Master kit. The hPCFT, NRF-1, Sp1, and KLF15 transcript levels were normalized to transcript levels for GAPDH. The scatter plots show univariate associations between hPCFT and NRF-1 ( A ), hPCFT and Sp1 ( B ), and hPCFT and KLF15 ( C ) by Pearson’s correlation coefficients ( r ). The x-axis and y-axis scales are logarithmic and the solid gray lines represent the fitted regression lines. N indicates the sample size (53). ( D ]. For the schematic shown, the thickness of the edge and the corresponding numbers (which range from 0 to 1) represent the relative strength of the association. The R/Bioconductor packages minet and igraph ].

    Article Snippet: The qPCRs were performed using a Roche LightCycler 480 and LightCycler 480 SYBR Green I Master Kit Mix (Roche Diagnostics), with primers located in the hPCFT core promoter region that includes putative binding sites for NRF-1, KLF15 or Sp1.

    Techniques: Expressing, Quantitative RT-PCR, SYBR Green Assay