biotinylated transcripts Search Results


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  • 93
    Thermo Fisher biotinylated
    Chordin affinity columns bind <t>biotinylated</t> cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.
    Biotinylated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore biotinylated rna transcripts
    Chordin affinity columns bind <t>biotinylated</t> cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.
    Biotinylated Rna Transcripts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher biotinylated transcripts
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated Transcripts, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Promega biotinylated transcripts
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated Transcripts, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneWorks biotinylated
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated, supplied by GeneWorks, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Enzo Biochem biotinylated nucleotides
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated Nucleotides, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem biotinylated ribonucleotide
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated Ribonucleotide, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 85/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem biotinylated utp
    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA <t>biotinylated</t> RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p
    Biotinylated Utp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche biotinylated utp
    SIRT1 binds directly to EV71 5′UTR, but not 3′UTR. (A–C) Cell extracts of RD, 293T or SK-N-SH cells were prepared and used as inputs, or incubated with no RNA, <t>biotin-16-UTP,</t> <t>non-biotinylated</t> EV71 5′UTR or biotinylated EV71 5′UTR (A). RD cell lysates were prepared and used as input, or were incubated with nonbiotinylated EV71 3′UTR RNA or biotinylated EV71 3′UTR RNA (B). RD cell lysates were prepared and used as input, or were incubated with biotinylated EV71 3′UTR RNA along with different concentrations of non-biotinylated EV71 3′UTR RNA or nonbiotinylated yeast tRNA (C). Protein–RNA pulldown assays were carried out with anti-SIRT1 antibody and precipitated with protein G. Interactions between SIRT1 and EV71 5′UTR were determined by western blotting with anti-SIRT1 antibody. (D) RD cells were infected with EV71 at an MOI of 10 for 12 h. Cell extracts were prepared and used for mRNA RNA extraction (Total RNA), or were used for protein–RNA pulldown assays with anti-SIRT1 antibody, anti-Flag antibody, without antibody or with water and followed by mRNA extraction. Then standard RT-PCR analysis using primers specific to EV71 5′UTR RNA or ribosomal protein S16 (RPS16) was performed.
    Biotinylated Utp, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson biotinylated abs
    Neuronal and Neural Crest Genes Expressed by the DP and Hair Follicles (A) Semi-quantitative RT-PCRs were conducted as in the legend to Figure 5 , except in this case, we used oligonucleotides against neuronal and/or neural crest expressed genes. In all cases, these genes either appeared on the DP molecular signature or scored as expressed by the DP as well as one or more of the other four skin populations. (see Figure 4 ) Shown are representative RT-PCR data, which show an excellent correlation with the DP-preferred expression pattern of the majority of these neural genes. (B) Immunohistochemistry and in situ hybridizations of neuronal/neural crest genes in skin. Sections of P4 backskins were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated <t>biotinylated</t> cRNA probes (sense controls were negative). Merged images of serial sections were used to compare Ab (red) and in situ (pseudogreen) patterns. Gfra1 , glial derived neurotrophic factor receptor1; Mdk , midkine; Prss12 , serine protease neurotrypsin; Tyr, tyrosinase. (C) Detection of neuronal/neural crest genes in highly enriched hair follicle preparations. Highly enriched follicle preparations were isolated by serial low-speed centrifugation following dispase and collagenase digestion of P4 backskins. After isolation and preparation of their mRNAs, semi-quantitative RT-PCR was conducted using oligonucleotides to those neuronal and neural crest markers that were found in the DP signature. As controls, oligonucleotides were used against Akp2 , Alx4 , Bmp6 , and Fgf7 , which are all markers that we mapped to DP by in situ hybridizations and/or immunofluorescence (see Figure 5 ). Note that the neuronal/neural crest genes appearing on the DP signature showed comparable signals to the documented DP genes.
    Biotinylated Abs, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc transcription 3 pstat3 igg
    Neuronal and Neural Crest Genes Expressed by the DP and Hair Follicles (A) Semi-quantitative RT-PCRs were conducted as in the legend to Figure 5 , except in this case, we used oligonucleotides against neuronal and/or neural crest expressed genes. In all cases, these genes either appeared on the DP molecular signature or scored as expressed by the DP as well as one or more of the other four skin populations. (see Figure 4 ) Shown are representative RT-PCR data, which show an excellent correlation with the DP-preferred expression pattern of the majority of these neural genes. (B) Immunohistochemistry and in situ hybridizations of neuronal/neural crest genes in skin. Sections of P4 backskins were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated <t>biotinylated</t> cRNA probes (sense controls were negative). Merged images of serial sections were used to compare Ab (red) and in situ (pseudogreen) patterns. Gfra1 , glial derived neurotrophic factor receptor1; Mdk , midkine; Prss12 , serine protease neurotrypsin; Tyr, tyrosinase. (C) Detection of neuronal/neural crest genes in highly enriched hair follicle preparations. Highly enriched follicle preparations were isolated by serial low-speed centrifugation following dispase and collagenase digestion of P4 backskins. After isolation and preparation of their mRNAs, semi-quantitative RT-PCR was conducted using oligonucleotides to those neuronal and neural crest markers that were found in the DP signature. As controls, oligonucleotides were used against Akp2 , Alx4 , Bmp6 , and Fgf7 , which are all markers that we mapped to DP by in situ hybridizations and/or immunofluorescence (see Figure 5 ). Note that the neuronal/neural crest genes appearing on the DP signature showed comparable signals to the documented DP genes.
    Transcription 3 Pstat3 Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc biotinylated protein ladder
    Characterization of spermatogenesis in Stag3 ko/ko mice Testis samples from wt ( Stag3 +/+ ) and Stag3 ko/ko mice, 40 days of age. RT-PCR analysis of testis mRNA from wt ( Stag3 +/+ ) and Stag3 ko/ko mice. The primer pairs are shown indicating the respective exons (E3, E4, E5); the expected size (bp) of the PCR products is provided. Immunoblot of testis nuclear extracts of the indicated mice, probed with anti-STAG3 or anti-SMC3 antibody as indicated. The anti-STAG3 antibody recognizes a specific band corresponding to the predicted molecular weight (141 kDa) of STAG3, which was present in wt but absent in Stag3 ko/ko extracts. An unspecific band is marked by an asterisk. A gel was loaded in parallel using the same extracts, and the corresponding membrane was probed with an antibody directed against SMC3, which has the same predicted molecular weight (141 kDa). The pictures are representative of three independent experiments. M = <t>biotinylated</t> protein marker. Immunofluorescence staining of spermatocyte chromosome spreads of wt and Stag3 ko/ko mice, probed with anti-SYCP3 antibody for AEs and SCs and anti-STAG3; nucleic acids were stained with DAPI. The stages of wt prophase I spermatocytes are indicated, and two examples of Stag3 ko/ko chromosome spreads are provided. Size bars indicate 10 μm. Source data are available online for this figure.
    Biotinylated Protein Ladder, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 455 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery 5 biotinylated derivatives
    Characterization of spermatogenesis in Stag3 ko/ko mice Testis samples from wt ( Stag3 +/+ ) and Stag3 ko/ko mice, 40 days of age. RT-PCR analysis of testis mRNA from wt ( Stag3 +/+ ) and Stag3 ko/ko mice. The primer pairs are shown indicating the respective exons (E3, E4, E5); the expected size (bp) of the PCR products is provided. Immunoblot of testis nuclear extracts of the indicated mice, probed with anti-STAG3 or anti-SMC3 antibody as indicated. The anti-STAG3 antibody recognizes a specific band corresponding to the predicted molecular weight (141 kDa) of STAG3, which was present in wt but absent in Stag3 ko/ko extracts. An unspecific band is marked by an asterisk. A gel was loaded in parallel using the same extracts, and the corresponding membrane was probed with an antibody directed against SMC3, which has the same predicted molecular weight (141 kDa). The pictures are representative of three independent experiments. M = <t>biotinylated</t> protein marker. Immunofluorescence staining of spermatocyte chromosome spreads of wt and Stag3 ko/ko mice, probed with anti-SYCP3 antibody for AEs and SCs and anti-STAG3; nucleic acids were stained with DAPI. The stages of wt prophase I spermatocytes are indicated, and two examples of Stag3 ko/ko chromosome spreads are provided. Size bars indicate 10 μm. Source data are available online for this figure.
    5 Biotinylated Derivatives, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare biotinylated
    Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing <t>biotinylated</t> GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.
    Biotinylated, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Horizon Discovery 5 biotinylated mrna
    Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing <t>biotinylated</t> GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.
    5 Biotinylated Mrna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher biotinylated egf
    FBP17 is required for endocytosis of EGFR. (A) Myc-tagged wild-type FBP17, SH3 domain–deleted FBP17, and 1–56 aa–deleted FBP17 were transfected in COS-7 cells. After starvation for 16 h, the transfected cells were incubated with Texas <t>red–EGF</t> (red) for 15 min, fixed, and stained with anti-Myc antibody (green). The percentage of cells with internalized EGF among FBP17-overexpressing cells was also shown with SD. Bar, 20 μm. (B) Quantitative EGF internalization assay of FBP17-transfected cells. The histogram shows uptake of <t>biotinylated</t> EGF as a function of total bound biotinylated EGF at indicated times in transfected cells. Data from three independent experiments. Error bars represent SD. (C) A431cells were transfected with the control, FBP17, CIP4, and Toca-1siRNA. After 24 h, a second transfection was performed and the cells were cultured for an additional 72 h and subjected to RT-PCR and Western blotting. (D) After 96 h, the cells transfected with the siRNA were incubated with Texas red-EGF for 10 min, fixed, and stained with anti-CIP4 antibody. (E) Quantitative EGF internalization assay of siRNA-transfected cells as in B. Data from three independent experiments. All error bars indicate SEM.
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    Biogenex biotinylated antidigoxigenin ab
    FBP17 is required for endocytosis of EGFR. (A) Myc-tagged wild-type FBP17, SH3 domain–deleted FBP17, and 1–56 aa–deleted FBP17 were transfected in COS-7 cells. After starvation for 16 h, the transfected cells were incubated with Texas <t>red–EGF</t> (red) for 15 min, fixed, and stained with anti-Myc antibody (green). The percentage of cells with internalized EGF among FBP17-overexpressing cells was also shown with SD. Bar, 20 μm. (B) Quantitative EGF internalization assay of FBP17-transfected cells. The histogram shows uptake of <t>biotinylated</t> EGF as a function of total bound biotinylated EGF at indicated times in transfected cells. Data from three independent experiments. Error bars represent SD. (C) A431cells were transfected with the control, FBP17, CIP4, and Toca-1siRNA. After 24 h, a second transfection was performed and the cells were cultured for an additional 72 h and subjected to RT-PCR and Western blotting. (D) After 96 h, the cells transfected with the siRNA were incubated with Texas red-EGF for 10 min, fixed, and stained with anti-CIP4 antibody. (E) Quantitative EGF internalization assay of siRNA-transfected cells as in B. Data from three independent experiments. All error bars indicate SEM.
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    Pharmingen biotinylated isotype specific control
    The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with <t>biotinylated</t> anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).
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    Nugen biotinylated nucleotide
    The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with <t>biotinylated</t> anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).
    Biotinylated Nucleotide, supplied by Nugen, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega biotinylated lys
    The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with <t>biotinylated</t> anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).
    Biotinylated Lys, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega 224 mer biotinylated utp
    The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with <t>biotinylated</t> anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).
    224 Mer Biotinylated Utp, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies biotinylated probe
    Receiver operating characteristic curves for head and neck squamous cell carcinomas by combined p16 immuno-histochemistry and HPV DNA testing by in-situ hybridization using the INFORM ® HPV-III Fam16(B) (Ventana, CA) and HPV16/18 <t>biotinylated</t> GenPoint ™ (Dako, CA) probes, or by MY-PCR, compared to HPV16 E6/E7 RT-PCR. Non-parametric receiver operating characteristic curves shown for combined test results with p16 immuno-histochemistry (assuming a 1+/≥10% cut-off) stratified by HPV Ventana in-situ hybridization (-■-), Dako in-situ hybridization (-▲-), or MY-PCR (-●-) results. Solitary markers shown reflect single test performance statistics for p16 IHC (assuming a 2+/≥75% cut-off) ( + ) and MY-PCR for HPV16 ( X ) alone. The dashed line indicates a reference test threshold with area under the receiver operating characteristic curve of 0.5.
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    Promega biotinylated oligo
    Validation of poly(A) fractionation microarray. T, total RNA; U, unbound; S, short (oligoadenylated RNA eluted with 0.075× SSC); L, long (polyadenylated RNA eluted with H 2 O). Gene names are according to Genbank, numbers in brackets indicate the corrected LogRatio obtained for the mRNA in the microarray screen. A high LogRatio should correlate with a short poly(A) tail. ( A ) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe and <t>biotinylated</t> <t>oligo(dT)</t> and then fractionated using the poly(A) fractionation method. Ten percent of the resulting fractions was analysed by urea–PAGE as in Figure 3 A. The lane with total RNA contains 2% of starting material. The remainder of the fractions with short and long poly(A) tails was used for microarray analysis (see Supplementary Table 2). ( B ) Total RNA from NIH3T3 was subjected to RNaseH treatment in the presence of oligo(dT) (+ lanes) or used untreated (− lanes) and subjected to RL-PAT using specific sense primers (Supplementary Table 1) for genes identified in the microarray analysis. The size difference in PCR products between RNaseH treated and untreated corresponds to the length of the poly(A) tail. The numbers after the gene name indicate the corrected LogRatio obtained in the microarray experiment. ( C ) Total RNA from NIH3T3 cells was treated with RNaseH in the presence of oligo(dT) and an antisense oligo specific for Actb (+) or the presence of the specific oligo only (−). The sample treated with the specific oligo only was subsequently fractionated using the poly(A) fractionation method (U, S, L). Actb RNA was detected by Northern analysis. ( D ) RNA was fractionated as described in (A). RT-PCR was performed on the resulting fractions. Samples in the no RT lane (−) were treated the same as samples in the total RNA lane (T) except for the omission of Superscript III. (E) RNA was fractionated as described in (A). Twenty-five percent of each fraction was then analysed by northern blotting. Total: 5% of starting material. The numbers in brackets behind the gene names refer to the average corrected LogRatio (see Supplementary Table 2).
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    Thermo Fisher secondary antimouse biotinylated antibody
    Validation of poly(A) fractionation microarray. T, total RNA; U, unbound; S, short (oligoadenylated RNA eluted with 0.075× SSC); L, long (polyadenylated RNA eluted with H 2 O). Gene names are according to Genbank, numbers in brackets indicate the corrected LogRatio obtained for the mRNA in the microarray screen. A high LogRatio should correlate with a short poly(A) tail. ( A ) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe and <t>biotinylated</t> <t>oligo(dT)</t> and then fractionated using the poly(A) fractionation method. Ten percent of the resulting fractions was analysed by urea–PAGE as in Figure 3 A. The lane with total RNA contains 2% of starting material. The remainder of the fractions with short and long poly(A) tails was used for microarray analysis (see Supplementary Table 2). ( B ) Total RNA from NIH3T3 was subjected to RNaseH treatment in the presence of oligo(dT) (+ lanes) or used untreated (− lanes) and subjected to RL-PAT using specific sense primers (Supplementary Table 1) for genes identified in the microarray analysis. The size difference in PCR products between RNaseH treated and untreated corresponds to the length of the poly(A) tail. The numbers after the gene name indicate the corrected LogRatio obtained in the microarray experiment. ( C ) Total RNA from NIH3T3 cells was treated with RNaseH in the presence of oligo(dT) and an antisense oligo specific for Actb (+) or the presence of the specific oligo only (−). The sample treated with the specific oligo only was subsequently fractionated using the poly(A) fractionation method (U, S, L). Actb RNA was detected by Northern analysis. ( D ) RNA was fractionated as described in (A). RT-PCR was performed on the resulting fractions. Samples in the no RT lane (−) were treated the same as samples in the total RNA lane (T) except for the omission of Superscript III. (E) RNA was fractionated as described in (A). Twenty-five percent of each fraction was then analysed by northern blotting. Total: 5% of starting material. The numbers in brackets behind the gene names refer to the average corrected LogRatio (see Supplementary Table 2).
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    Image Search Results


    Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

    Journal: EMBO Reports

    Article Title: Integrin-?3 mediates binding of Chordin to the cell surface and promotes its endocytosis

    doi: 10.1038/sj.embor.embor902

    Figure Lengend Snippet: Chordin affinity columns bind biotinylated cell-surface Integrin-α3. ( A ) A Chordin (Chd)–Fc affinity matrix binds two distinct cell-surface proteins in COS-7 cells. Cell extracts containing biotinylated surface proteins from COS-7 cells were bound to secreted Fc (s-Fc; lane 2) or Chd–Fc (lane 3) columns, or were immunoprecipitated using an anti-Integrin-α3 (Int-α3) antibody (lane 5). Proteins bound to the columns were analysed by immunoblotting with streptavidin–horseradish-peroxidase (SA–HRP; Pierce). Lane 1 shows loading of 1% of the total biotinylated cell lysate. ( B ) A Chordin affinity matrix binds Integrin-α3. COS-7 cell extracts were bound to s-Fc (lane 2) or Chd–Fc affinity columns (lane 3), eluted, and analysed by immunoblotting with anti-Integrin-α3. ( C – G ) Chordin and Integrin-α3 are co-expressed during embryonic development. In situ hybridization analysis of chordin ( C , D ) and integrin-a3 ( E , F ) expression at stages 10.5 and 32. ( G ) RT–PCR (PCR after reverse transcription) analysis of chordin and integrin-a3 messenger RNA levels in control and LiCl-treated embryos. Elongation factor-1α (EF-1α) was used as a loading control.

    Article Snippet: Cell-surface proteins from COS-7 cells were biotinylated (using EZ-link-Biotin; Pierce), solubilized with 0.1% Triton X-100 (as in ), and loaded on Chd–Fc or s-Fc affinity columns.

    Techniques: Immunoprecipitation, In Situ Hybridization, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA biotinylated RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p

    Journal: Nature Communications

    Article Title: TERRA recruitment of polycomb to telomeres is essential for histone trymethylation marks at telomeric heterochromatin

    doi: 10.1038/s41467-018-03916-3

    Figure Lengend Snippet: TERRAs bind PRC2 and modulates its own and HP1 recruitment to telomeres. a A TERRA biotinylated RNA oligo (S) was incubated with nuclear extracts from U2OS cells and their association with Ezh2 and SUZ12 was detected by western blotting. A biotinylated control RNA oligo corresponding to the complementary sequence (AS) of the same length as the biotinylated TERRA (N 48 ) was used as control. Biotin pull-down in the absence of RNA oligo (no oligo) was included to monitor inespecific binding to the beads. b Representative images of the average number of colocalizations found on double immunostaining to TRF2 (green) and SUZ12 (red) in the U2OS WT and 20q-KO clones. Arrowheads indicate colocalization events. Scale bar, 10 μm. (Left graph) Quantification of the colocalization in each of the WT and 20q-TERRA KO clones (mean values ± s.e.m., n = number of cells) and (right graph) in all WT vs. the 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). c Telomeric ChIP-dot-blot of SUZ12 in U2OS cells infected with scramble or SUZ12 shRNA. IgG was used as a control. DNA input is also shown. (Graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input (mean values ± s.e.m., n = technical triplicates). d Representative confocal STED super-resolution images showing the colocalization between TRF2 (in green) and SUZ12 (in red) in U2OS cells. Zoom: a colocalization event. Scale bar 5 μm. e Telomeric ChIP-dot-blot for HP1 in WT and 20q-TERRA KO clones. IgG was used as a control. DNA input signal is also shown. (Left graph) Quantification of the signal from the immunoprecipitated telomeric repeats normalized by the input for each individual sample and (right graph) for all WT vs. 20q-TERRA KO clones (mean values ± s.e.m., n = independent clone). Student’s t -test was used for statistical analysis (* p

    Article Snippet: Biotin pull-down analysis Biotin pull-down assays were carried out as described in ref. except for that 150 μg of nuclear lysate were incubated with 0.9 ng of biotinylated transcripts (Invitrogen) for 1 h at room temperature.

    Techniques: Incubation, Western Blot, Sequencing, Binding Assay, Double Immunostaining, Clone Assay, Chromatin Immunoprecipitation, Dot Blot, Infection, shRNA, Immunoprecipitation

    SIRT1 binds directly to EV71 5′UTR, but not 3′UTR. (A–C) Cell extracts of RD, 293T or SK-N-SH cells were prepared and used as inputs, or incubated with no RNA, biotin-16-UTP, non-biotinylated EV71 5′UTR or biotinylated EV71 5′UTR (A). RD cell lysates were prepared and used as input, or were incubated with nonbiotinylated EV71 3′UTR RNA or biotinylated EV71 3′UTR RNA (B). RD cell lysates were prepared and used as input, or were incubated with biotinylated EV71 3′UTR RNA along with different concentrations of non-biotinylated EV71 3′UTR RNA or nonbiotinylated yeast tRNA (C). Protein–RNA pulldown assays were carried out with anti-SIRT1 antibody and precipitated with protein G. Interactions between SIRT1 and EV71 5′UTR were determined by western blotting with anti-SIRT1 antibody. (D) RD cells were infected with EV71 at an MOI of 10 for 12 h. Cell extracts were prepared and used for mRNA RNA extraction (Total RNA), or were used for protein–RNA pulldown assays with anti-SIRT1 antibody, anti-Flag antibody, without antibody or with water and followed by mRNA extraction. Then standard RT-PCR analysis using primers specific to EV71 5′UTR RNA or ribosomal protein S16 (RPS16) was performed.

    Journal: Journal of Cell Science

    Article Title: SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

    doi: 10.1242/jcs.193698

    Figure Lengend Snippet: SIRT1 binds directly to EV71 5′UTR, but not 3′UTR. (A–C) Cell extracts of RD, 293T or SK-N-SH cells were prepared and used as inputs, or incubated with no RNA, biotin-16-UTP, non-biotinylated EV71 5′UTR or biotinylated EV71 5′UTR (A). RD cell lysates were prepared and used as input, or were incubated with nonbiotinylated EV71 3′UTR RNA or biotinylated EV71 3′UTR RNA (B). RD cell lysates were prepared and used as input, or were incubated with biotinylated EV71 3′UTR RNA along with different concentrations of non-biotinylated EV71 3′UTR RNA or nonbiotinylated yeast tRNA (C). Protein–RNA pulldown assays were carried out with anti-SIRT1 antibody and precipitated with protein G. Interactions between SIRT1 and EV71 5′UTR were determined by western blotting with anti-SIRT1 antibody. (D) RD cells were infected with EV71 at an MOI of 10 for 12 h. Cell extracts were prepared and used for mRNA RNA extraction (Total RNA), or were used for protein–RNA pulldown assays with anti-SIRT1 antibody, anti-Flag antibody, without antibody or with water and followed by mRNA extraction. Then standard RT-PCR analysis using primers specific to EV71 5′UTR RNA or ribosomal protein S16 (RPS16) was performed.

    Article Snippet: Biotinylated RNA was synthesized in 20 μl transcription reaction mixtures containing 0.5 μl 20 mM biotinylated UTP [biotin-16-UTP; Roche].

    Techniques: Incubation, Western Blot, Infection, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    Neuronal and Neural Crest Genes Expressed by the DP and Hair Follicles (A) Semi-quantitative RT-PCRs were conducted as in the legend to Figure 5 , except in this case, we used oligonucleotides against neuronal and/or neural crest expressed genes. In all cases, these genes either appeared on the DP molecular signature or scored as expressed by the DP as well as one or more of the other four skin populations. (see Figure 4 ) Shown are representative RT-PCR data, which show an excellent correlation with the DP-preferred expression pattern of the majority of these neural genes. (B) Immunohistochemistry and in situ hybridizations of neuronal/neural crest genes in skin. Sections of P4 backskins were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative). Merged images of serial sections were used to compare Ab (red) and in situ (pseudogreen) patterns. Gfra1 , glial derived neurotrophic factor receptor1; Mdk , midkine; Prss12 , serine protease neurotrypsin; Tyr, tyrosinase. (C) Detection of neuronal/neural crest genes in highly enriched hair follicle preparations. Highly enriched follicle preparations were isolated by serial low-speed centrifugation following dispase and collagenase digestion of P4 backskins. After isolation and preparation of their mRNAs, semi-quantitative RT-PCR was conducted using oligonucleotides to those neuronal and neural crest markers that were found in the DP signature. As controls, oligonucleotides were used against Akp2 , Alx4 , Bmp6 , and Fgf7 , which are all markers that we mapped to DP by in situ hybridizations and/or immunofluorescence (see Figure 5 ). Note that the neuronal/neural crest genes appearing on the DP signature showed comparable signals to the documented DP genes.

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Neuronal and Neural Crest Genes Expressed by the DP and Hair Follicles (A) Semi-quantitative RT-PCRs were conducted as in the legend to Figure 5 , except in this case, we used oligonucleotides against neuronal and/or neural crest expressed genes. In all cases, these genes either appeared on the DP molecular signature or scored as expressed by the DP as well as one or more of the other four skin populations. (see Figure 4 ) Shown are representative RT-PCR data, which show an excellent correlation with the DP-preferred expression pattern of the majority of these neural genes. (B) Immunohistochemistry and in situ hybridizations of neuronal/neural crest genes in skin. Sections of P4 backskins were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative). Merged images of serial sections were used to compare Ab (red) and in situ (pseudogreen) patterns. Gfra1 , glial derived neurotrophic factor receptor1; Mdk , midkine; Prss12 , serine protease neurotrypsin; Tyr, tyrosinase. (C) Detection of neuronal/neural crest genes in highly enriched hair follicle preparations. Highly enriched follicle preparations were isolated by serial low-speed centrifugation following dispase and collagenase digestion of P4 backskins. After isolation and preparation of their mRNAs, semi-quantitative RT-PCR was conducted using oligonucleotides to those neuronal and neural crest markers that were found in the DP signature. As controls, oligonucleotides were used against Akp2 , Alx4 , Bmp6 , and Fgf7 , which are all markers that we mapped to DP by in situ hybridizations and/or immunofluorescence (see Figure 5 ). Note that the neuronal/neural crest genes appearing on the DP signature showed comparable signals to the documented DP genes.

    Article Snippet: DP were obtained after first depleting Mc (CD117+ ), lymphocytes (CD45+ ), and endothelial cells (CD34+ ) with biotinylated Abs (BD Pharmingen, San Diego, California, United States)/magnetic anti-biotin microbeads (Miltenyi Biotec, Bergish Gladbach, Germany), and then selecting for RFPhigh GFP− cells in the FACS.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemistry, In Situ, Immunofluorescence, In Situ Hybridization, Derivative Assay, Isolation, Centrifugation, Quantitative RT-PCR

    Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Article Snippet: DP were obtained after first depleting Mc (CD117+ ), lymphocytes (CD45+ ), and endothelial cells (CD34+ ) with biotinylated Abs (BD Pharmingen, San Diego, California, United States)/magnetic anti-biotin microbeads (Miltenyi Biotec, Bergish Gladbach, Germany), and then selecting for RFPhigh GFP− cells in the FACS.

    Techniques: Quantitative RT-PCR, Isolation, Expressing, Transduction, Agarose Gel Electrophoresis, Immunohistochemistry, In Situ, Mouse Assay, Immunofluorescence, In Situ Hybridization

    Characterization of spermatogenesis in Stag3 ko/ko mice Testis samples from wt ( Stag3 +/+ ) and Stag3 ko/ko mice, 40 days of age. RT-PCR analysis of testis mRNA from wt ( Stag3 +/+ ) and Stag3 ko/ko mice. The primer pairs are shown indicating the respective exons (E3, E4, E5); the expected size (bp) of the PCR products is provided. Immunoblot of testis nuclear extracts of the indicated mice, probed with anti-STAG3 or anti-SMC3 antibody as indicated. The anti-STAG3 antibody recognizes a specific band corresponding to the predicted molecular weight (141 kDa) of STAG3, which was present in wt but absent in Stag3 ko/ko extracts. An unspecific band is marked by an asterisk. A gel was loaded in parallel using the same extracts, and the corresponding membrane was probed with an antibody directed against SMC3, which has the same predicted molecular weight (141 kDa). The pictures are representative of three independent experiments. M = biotinylated protein marker. Immunofluorescence staining of spermatocyte chromosome spreads of wt and Stag3 ko/ko mice, probed with anti-SYCP3 antibody for AEs and SCs and anti-STAG3; nucleic acids were stained with DAPI. The stages of wt prophase I spermatocytes are indicated, and two examples of Stag3 ko/ko chromosome spreads are provided. Size bars indicate 10 μm. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion

    doi: 10.1002/embj.201387330

    Figure Lengend Snippet: Characterization of spermatogenesis in Stag3 ko/ko mice Testis samples from wt ( Stag3 +/+ ) and Stag3 ko/ko mice, 40 days of age. RT-PCR analysis of testis mRNA from wt ( Stag3 +/+ ) and Stag3 ko/ko mice. The primer pairs are shown indicating the respective exons (E3, E4, E5); the expected size (bp) of the PCR products is provided. Immunoblot of testis nuclear extracts of the indicated mice, probed with anti-STAG3 or anti-SMC3 antibody as indicated. The anti-STAG3 antibody recognizes a specific band corresponding to the predicted molecular weight (141 kDa) of STAG3, which was present in wt but absent in Stag3 ko/ko extracts. An unspecific band is marked by an asterisk. A gel was loaded in parallel using the same extracts, and the corresponding membrane was probed with an antibody directed against SMC3, which has the same predicted molecular weight (141 kDa). The pictures are representative of three independent experiments. M = biotinylated protein marker. Immunofluorescence staining of spermatocyte chromosome spreads of wt and Stag3 ko/ko mice, probed with anti-SYCP3 antibody for AEs and SCs and anti-STAG3; nucleic acids were stained with DAPI. The stages of wt prophase I spermatocytes are indicated, and two examples of Stag3 ko/ko chromosome spreads are provided. Size bars indicate 10 μm. Source data are available online for this figure.

    Article Snippet: A biotinylated protein ladder (Cell Signaling Technology 7727) was loaded onto each gel and detected using an anti-biotin HRP (Cell Signaling Technology 7075).

    Techniques: Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Molecular Weight, Marker, Immunofluorescence, Staining

    Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.

    Journal: Nucleic Acids Research

    Article Title: Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

    doi: 10.1093/nar/gni080

    Figure Lengend Snippet: Specificity of selected anti-GAPDH antibody fragments determined by ELISA. Characterization of phage and soluble Fab anti-GAPDH antibodies by ELISA. The assay was performed by immobilizing biotinylated GAPDH on a polystyrene plate. Phage-displayed antibodies reactive with the coated antigen were detected with peroxidase-conjugated anti-M13 antibody (Amersham), while the detection of soluble Fabs was performed using 9E10 anti-c-myc monoclonal antibody (final concentration 2 μg/ml) followed by peroxidase-conjugated rabbit anti-mouse antibody (1:1000). The results of the assay are shown as absorbance at 450 nm. In the case of the data obtained with the phage preparation and the sFab cell supernatant, results were not normalized for protein concentrations.

    Article Snippet: All biotinylated products were purified from an agarose gel with the GFX extraction kit (Amersham).

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Phage Preparation

    The ONCL technology. ( A ) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. ( B ) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.

    Journal: Nucleic Acids Research

    Article Title: Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

    doi: 10.1093/nar/gni080

    Figure Lengend Snippet: The ONCL technology. ( A ) Schematic outline of the ONCL technology. RACE: decapped mRNA is ligated to a RNA adapter, then converted to cDNA by RT–PCR. cDNA is then amplified using a 5′-biotinylated primer complementary to the adapter sequence combined with a 3′-primer either complementary to the human IgG-derived heavy chain constant region or to the light chain constant regions. DNA immobilization: double-stranded (ds) biotinylated RACE-derived PCR products are bound to streptavidin-coupled magnetic beads. After the DNA–beads complex is immobilized on a magnetic stand, ssDNA is prepared by NaOH denaturation. Cleavage of ssDNA: annealing of the adapter oligonucleotides to the top strand retained on the beads creates a dsDNA region accessible for the restriction enzyme (E1). Cleaved single-stranded V gene is released from the beads and can now be used in the next step. Preparation of the V-gene ssDNA for cloning: The ssDNA is then ligated to a 100× excess of partially dsDNA made through the annealing of two oligonucleotides. The lower-strand tail of this oligonucleotide duplex is complementary to the 5′ end of the ssDNA, allowing their recognition during the ligation procedure. The ligated product is then amplified using primers appended with appropriate restriction sites (plain black arrows), allowing the directional cloning of the V genes into the phagemid vector. E represents restriction enzymes, vertical rectangles represent restriction sites, asterisks represent biotin, dark rectangles represent the tailed sequence and ‘base-adorned’ arrows represent base pairing of the oligonucleotides with the ssDNA. ( B ) Example of ONCL method: λ1 V-gene capture and cloning. (1) Annealing of adapter for family Vλ1 to Vλ1 genes, immobilized on beads—cleavage by HinfI; (2) release of cleaved Vλ1 genes in supernatant; (3) annealing and ligation of cleaved Vλ1 genes to hybridized Vλ1 bridge and extender (ApaLI site underscored); (4) amplification of ligated DNA using ONPlePCR and Cλ2,7forAsc primers; (5) directional cloning into pMid21 of Vλ1 genes via ApaLI and AscI. X, non-V-gene related amino acid sequence.

    Article Snippet: All biotinylated products were purified from an agarose gel with the GFX extraction kit (Amersham).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Polymerase Chain Reaction, Magnetic Beads, Clone Assay, Ligation, Plasmid Preparation

    FBP17 is required for endocytosis of EGFR. (A) Myc-tagged wild-type FBP17, SH3 domain–deleted FBP17, and 1–56 aa–deleted FBP17 were transfected in COS-7 cells. After starvation for 16 h, the transfected cells were incubated with Texas red–EGF (red) for 15 min, fixed, and stained with anti-Myc antibody (green). The percentage of cells with internalized EGF among FBP17-overexpressing cells was also shown with SD. Bar, 20 μm. (B) Quantitative EGF internalization assay of FBP17-transfected cells. The histogram shows uptake of biotinylated EGF as a function of total bound biotinylated EGF at indicated times in transfected cells. Data from three independent experiments. Error bars represent SD. (C) A431cells were transfected with the control, FBP17, CIP4, and Toca-1siRNA. After 24 h, a second transfection was performed and the cells were cultured for an additional 72 h and subjected to RT-PCR and Western blotting. (D) After 96 h, the cells transfected with the siRNA were incubated with Texas red-EGF for 10 min, fixed, and stained with anti-CIP4 antibody. (E) Quantitative EGF internalization assay of siRNA-transfected cells as in B. Data from three independent experiments. All error bars indicate SEM.

    Journal: The Journal of Cell Biology

    Article Title: Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis

    doi: 10.1083/jcb.200508091

    Figure Lengend Snippet: FBP17 is required for endocytosis of EGFR. (A) Myc-tagged wild-type FBP17, SH3 domain–deleted FBP17, and 1–56 aa–deleted FBP17 were transfected in COS-7 cells. After starvation for 16 h, the transfected cells were incubated with Texas red–EGF (red) for 15 min, fixed, and stained with anti-Myc antibody (green). The percentage of cells with internalized EGF among FBP17-overexpressing cells was also shown with SD. Bar, 20 μm. (B) Quantitative EGF internalization assay of FBP17-transfected cells. The histogram shows uptake of biotinylated EGF as a function of total bound biotinylated EGF at indicated times in transfected cells. Data from three independent experiments. Error bars represent SD. (C) A431cells were transfected with the control, FBP17, CIP4, and Toca-1siRNA. After 24 h, a second transfection was performed and the cells were cultured for an additional 72 h and subjected to RT-PCR and Western blotting. (D) After 96 h, the cells transfected with the siRNA were incubated with Texas red-EGF for 10 min, fixed, and stained with anti-CIP4 antibody. (E) Quantitative EGF internalization assay of siRNA-transfected cells as in B. Data from three independent experiments. All error bars indicate SEM.

    Article Snippet: After 72 h, cells were starved with serum-free DME for 16 h. Cells were then incubated with 20 ng/ml of biotinylated EGF (Invitrogen) for 30 min at 4°C and moved to a 37°C incubator for the indicated times.

    Techniques: Transfection, Incubation, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot

    The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with biotinylated anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).

    Journal: The Korean Journal of Parasitology

    Article Title: Sequential analysis of cell differentials and IFN-? production of splenocytes from mice infected with Toxoplasma gondii

    doi: 10.3347/kjp.2000.38.2.85

    Figure Lengend Snippet: The kinetics of IFN-γ mRNA expression of CD3 - and CD3 + cells after Toxoplasma gondii infection. A. Flow cytometric profile of CD3 - and CD3 + cells. Splenocytes were stained first with biotinylated anti-mouse CD3 mAb, followed by streptavidin-FITC, and then analyzed by FACScan. M1, CD3 + cells; M2, CD3 - cells. B. Relative IFN-γ mRNA experssion level of splenic CD3 - and CD3 + cells. The mRNA expression for IFN-γ was assayed using RT-PCR. The differences in the transcriptional level for all the genes were expressed relative to the uninfected mice (assigned as 1).

    Article Snippet: Briefly, splenocytes containing 1×106 cells were incubated with either 50 µl of biotinylated anti-mouse CD3 mAb or biotinylated isotype-specific control (PharMingen) for 60 min at 4℃.

    Techniques: Expressing, Infection, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    Receiver operating characteristic curves for head and neck squamous cell carcinomas by combined p16 immuno-histochemistry and HPV DNA testing by in-situ hybridization using the INFORM ® HPV-III Fam16(B) (Ventana, CA) and HPV16/18 biotinylated GenPoint ™ (Dako, CA) probes, or by MY-PCR, compared to HPV16 E6/E7 RT-PCR. Non-parametric receiver operating characteristic curves shown for combined test results with p16 immuno-histochemistry (assuming a 1+/≥10% cut-off) stratified by HPV Ventana in-situ hybridization (-■-), Dako in-situ hybridization (-▲-), or MY-PCR (-●-) results. Solitary markers shown reflect single test performance statistics for p16 IHC (assuming a 2+/≥75% cut-off) ( + ) and MY-PCR for HPV16 ( X ) alone. The dashed line indicates a reference test threshold with area under the receiver operating characteristic curve of 0.5.

    Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

    Article Title: A comparison of clinically utilized human papillomavirus detection methods in head and neck cancer

    doi: 10.1038/modpathol.2011.91

    Figure Lengend Snippet: Receiver operating characteristic curves for head and neck squamous cell carcinomas by combined p16 immuno-histochemistry and HPV DNA testing by in-situ hybridization using the INFORM ® HPV-III Fam16(B) (Ventana, CA) and HPV16/18 biotinylated GenPoint ™ (Dako, CA) probes, or by MY-PCR, compared to HPV16 E6/E7 RT-PCR. Non-parametric receiver operating characteristic curves shown for combined test results with p16 immuno-histochemistry (assuming a 1+/≥10% cut-off) stratified by HPV Ventana in-situ hybridization (-■-), Dako in-situ hybridization (-▲-), or MY-PCR (-●-) results. Solitary markers shown reflect single test performance statistics for p16 IHC (assuming a 2+/≥75% cut-off) ( + ) and MY-PCR for HPV16 ( X ) alone. The dashed line indicates a reference test threshold with area under the receiver operating characteristic curve of 0.5.

    Article Snippet: One hundred-and-ten prospectively collected, formalin fixed tumor specimens were compiled onto tissue microarrays and tested for human papillomavirus DNA by in-situ hybridization with two probe sets: a biotinylated probe for high-risk human papillomavirus types 16/18 (Dako, CA), and a probe cocktail for 16/18 plus 10 additional high-risk types (Ventana, AZ).

    Techniques: Immunohistochemistry, DNA In Situ Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization

    Validation of poly(A) fractionation microarray. T, total RNA; U, unbound; S, short (oligoadenylated RNA eluted with 0.075× SSC); L, long (polyadenylated RNA eluted with H 2 O). Gene names are according to Genbank, numbers in brackets indicate the corrected LogRatio obtained for the mRNA in the microarray screen. A high LogRatio should correlate with a short poly(A) tail. ( A ) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe and biotinylated oligo(dT) and then fractionated using the poly(A) fractionation method. Ten percent of the resulting fractions was analysed by urea–PAGE as in Figure 3 A. The lane with total RNA contains 2% of starting material. The remainder of the fractions with short and long poly(A) tails was used for microarray analysis (see Supplementary Table 2). ( B ) Total RNA from NIH3T3 was subjected to RNaseH treatment in the presence of oligo(dT) (+ lanes) or used untreated (− lanes) and subjected to RL-PAT using specific sense primers (Supplementary Table 1) for genes identified in the microarray analysis. The size difference in PCR products between RNaseH treated and untreated corresponds to the length of the poly(A) tail. The numbers after the gene name indicate the corrected LogRatio obtained in the microarray experiment. ( C ) Total RNA from NIH3T3 cells was treated with RNaseH in the presence of oligo(dT) and an antisense oligo specific for Actb (+) or the presence of the specific oligo only (−). The sample treated with the specific oligo only was subsequently fractionated using the poly(A) fractionation method (U, S, L). Actb RNA was detected by Northern analysis. ( D ) RNA was fractionated as described in (A). RT-PCR was performed on the resulting fractions. Samples in the no RT lane (−) were treated the same as samples in the total RNA lane (T) except for the omission of Superscript III. (E) RNA was fractionated as described in (A). Twenty-five percent of each fraction was then analysed by northern blotting. Total: 5% of starting material. The numbers in brackets behind the gene names refer to the average corrected LogRatio (see Supplementary Table 2).

    Journal: Nucleic Acids Research

    Article Title: A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells

    doi: 10.1093/nar/gkm830

    Figure Lengend Snippet: Validation of poly(A) fractionation microarray. T, total RNA; U, unbound; S, short (oligoadenylated RNA eluted with 0.075× SSC); L, long (polyadenylated RNA eluted with H 2 O). Gene names are according to Genbank, numbers in brackets indicate the corrected LogRatio obtained for the mRNA in the microarray screen. A high LogRatio should correlate with a short poly(A) tail. ( A ) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe and biotinylated oligo(dT) and then fractionated using the poly(A) fractionation method. Ten percent of the resulting fractions was analysed by urea–PAGE as in Figure 3 A. The lane with total RNA contains 2% of starting material. The remainder of the fractions with short and long poly(A) tails was used for microarray analysis (see Supplementary Table 2). ( B ) Total RNA from NIH3T3 was subjected to RNaseH treatment in the presence of oligo(dT) (+ lanes) or used untreated (− lanes) and subjected to RL-PAT using specific sense primers (Supplementary Table 1) for genes identified in the microarray analysis. The size difference in PCR products between RNaseH treated and untreated corresponds to the length of the poly(A) tail. The numbers after the gene name indicate the corrected LogRatio obtained in the microarray experiment. ( C ) Total RNA from NIH3T3 cells was treated with RNaseH in the presence of oligo(dT) and an antisense oligo specific for Actb (+) or the presence of the specific oligo only (−). The sample treated with the specific oligo only was subsequently fractionated using the poly(A) fractionation method (U, S, L). Actb RNA was detected by Northern analysis. ( D ) RNA was fractionated as described in (A). RT-PCR was performed on the resulting fractions. Samples in the no RT lane (−) were treated the same as samples in the total RNA lane (T) except for the omission of Superscript III. (E) RNA was fractionated as described in (A). Twenty-five percent of each fraction was then analysed by northern blotting. Total: 5% of starting material. The numbers in brackets behind the gene names refer to the average corrected LogRatio (see Supplementary Table 2).

    Article Snippet: Five microlitres polyadenylated probe mix (see above) and 10 μl (for oocytes) or 15 μl (for RNA from NIH3T3 cells) biotinylated oligo(dT) (50 pmol/μl, Promega) were added.

    Techniques: Fractionation, Microarray, Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Northern Blot, Reverse Transcription Polymerase Chain Reaction