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  • 99
    Millipore pretrox tight pur
    FLII is required for P-Rex1-Rac1-driven cell migration. ( a ) Western blot of NIH3T3 cells <t>(NIH3T3+pRetroX</t> P-Rex1 WT) treated with mock, NT or two different siRNAs against FLII (FLII siRNA1 and FLII siRNA2) in the presence of ethanol (−dox) or 1 μg ml −1 doxycycline (+dox) to induce expression of P-Rex1 WT. Levels of endogenous FLII and P-Rex1 expression were detected by western blot analysis. ( b ) Representative fluorescence images following Oris migration assay of cells described in a . Scale bar, 200 μm. ( c ) Quantification of cell migration of NIH3T3 cells described in a normalized to −dox treated mock cells. ( d – g ) Single-cell tracking of NIH3T3 cells expressing P-Rex1 WT following dox induction and treatment with indicated oligos. Graphs show the effect of FLII knockdown on ( d ) accumulated distance, ( e ) displacement, ( f ) speed and ( g ) straightness. ( h ) Western blot of CHL1 cells transfected with NT oligo or two different siRNAs against human FLII (hFLII siRNA1 and hFLII siRNA2). Levels of endogenous FLII were detected by western blot analysis. In a , h α-Tubulin was used as a loading control. Representative western blots from three independent experiments. ( i ) Representative fluorescence images following Oris migration assay of CHL1 cells treated with indicated oligos. Scale bar, 200 μm. ( j ) Quantification of cell migration of CHL1 cells described in h normalized to mock treated cells. For c , j graphs represent the average per cent migration±s.e.m. from three independent experiments. For c – g , j , Student's t -test was used to assess significance as indicated on graphs. P values indicated above each bar are relative to −dox treated mock cells ( c ), or NT control ( d – g ) or mock treated cells ( j ). NS=non-significant; *= P ≤0.05; **= P ≤0.01; ***= P ≤0.001.
    Pretrox Tight Pur, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pretrox tight pur
    Expression of dominant-negative Rac1 blocks the induction of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N) or both constructs, were generated using the <t>pRetroX-Tight-Pur</t> Tet-On system
    Pretrox Tight Pur, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa pretrox tight pur vector
    Expression of dominant-negative Rac1 blocks the induction of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N) or both constructs, were generated using the <t>pRetroX-Tight-Pur</t> Tet-On system
    Pretrox Tight Pur Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa pretrox tight pur retroviral vector
    Expression of dominant-negative Rac1 blocks the induction of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N) or both constructs, were generated using the <t>pRetroX-Tight-Pur</t> Tet-On system
    Pretrox Tight Pur Retroviral Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa pretrox tight pur luc plasmid
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Luc Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa pretrox tight pur retrovirus vector
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Retrovirus Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa expression vector pretrox tight pur
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Expression Vector Pretrox Tight Pur, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pretrox tight pur clontech cut
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Clontech Cut, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa pretrox tight pur luc
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Luc, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa doxycycline inducible retroviral vector pretrox tight pur
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Doxycycline Inducible Retroviral Vector Pretrox Tight Pur, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa pretrox tight pur response vector prxtpl
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Response Vector Prxtpl, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pretrox tet on advanced pretrox tight pur system
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tet On Advanced Pretrox Tight Pur System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa tetracycline inducible retroviral expression vector pretrox tight pur goi
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Tetracycline Inducible Retroviral Expression Vector Pretrox Tight Pur Goi, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pretrox tight pur clonetech doxycycline inducible vector
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pretrox Tight Pur Clonetech Doxycycline Inducible Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pqcxip
    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the <t>pRetroX-Tight-Pur-Luc</t> vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.
    Pqcxip, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FLII is required for P-Rex1-Rac1-driven cell migration. ( a ) Western blot of NIH3T3 cells (NIH3T3+pRetroX P-Rex1 WT) treated with mock, NT or two different siRNAs against FLII (FLII siRNA1 and FLII siRNA2) in the presence of ethanol (−dox) or 1 μg ml −1 doxycycline (+dox) to induce expression of P-Rex1 WT. Levels of endogenous FLII and P-Rex1 expression were detected by western blot analysis. ( b ) Representative fluorescence images following Oris migration assay of cells described in a . Scale bar, 200 μm. ( c ) Quantification of cell migration of NIH3T3 cells described in a normalized to −dox treated mock cells. ( d – g ) Single-cell tracking of NIH3T3 cells expressing P-Rex1 WT following dox induction and treatment with indicated oligos. Graphs show the effect of FLII knockdown on ( d ) accumulated distance, ( e ) displacement, ( f ) speed and ( g ) straightness. ( h ) Western blot of CHL1 cells transfected with NT oligo or two different siRNAs against human FLII (hFLII siRNA1 and hFLII siRNA2). Levels of endogenous FLII were detected by western blot analysis. In a , h α-Tubulin was used as a loading control. Representative western blots from three independent experiments. ( i ) Representative fluorescence images following Oris migration assay of CHL1 cells treated with indicated oligos. Scale bar, 200 μm. ( j ) Quantification of cell migration of CHL1 cells described in h normalized to mock treated cells. For c , j graphs represent the average per cent migration±s.e.m. from three independent experiments. For c – g , j , Student's t -test was used to assess significance as indicated on graphs. P values indicated above each bar are relative to −dox treated mock cells ( c ), or NT control ( d – g ) or mock treated cells ( j ). NS=non-significant; *= P ≤0.05; **= P ≤0.01; ***= P ≤0.001.

    Journal: Nature Communications

    Article Title: Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    doi: 10.1038/ncomms10664

    Figure Lengend Snippet: FLII is required for P-Rex1-Rac1-driven cell migration. ( a ) Western blot of NIH3T3 cells (NIH3T3+pRetroX P-Rex1 WT) treated with mock, NT or two different siRNAs against FLII (FLII siRNA1 and FLII siRNA2) in the presence of ethanol (−dox) or 1 μg ml −1 doxycycline (+dox) to induce expression of P-Rex1 WT. Levels of endogenous FLII and P-Rex1 expression were detected by western blot analysis. ( b ) Representative fluorescence images following Oris migration assay of cells described in a . Scale bar, 200 μm. ( c ) Quantification of cell migration of NIH3T3 cells described in a normalized to −dox treated mock cells. ( d – g ) Single-cell tracking of NIH3T3 cells expressing P-Rex1 WT following dox induction and treatment with indicated oligos. Graphs show the effect of FLII knockdown on ( d ) accumulated distance, ( e ) displacement, ( f ) speed and ( g ) straightness. ( h ) Western blot of CHL1 cells transfected with NT oligo or two different siRNAs against human FLII (hFLII siRNA1 and hFLII siRNA2). Levels of endogenous FLII were detected by western blot analysis. In a , h α-Tubulin was used as a loading control. Representative western blots from three independent experiments. ( i ) Representative fluorescence images following Oris migration assay of CHL1 cells treated with indicated oligos. Scale bar, 200 μm. ( j ) Quantification of cell migration of CHL1 cells described in h normalized to mock treated cells. For c , j graphs represent the average per cent migration±s.e.m. from three independent experiments. For c – g , j , Student's t -test was used to assess significance as indicated on graphs. P values indicated above each bar are relative to −dox treated mock cells ( c ), or NT control ( d – g ) or mock treated cells ( j ). NS=non-significant; *= P ≤0.05; **= P ≤0.01; ***= P ≤0.001.

    Article Snippet: Cells with both the pRetroX-Tet-On Advanced and pRetroX-Tight-Pur (EV or with indicated proteins) were selected using 1 mg ml−1 G418 and 2 μg ml−1 Puromycin (Sigma-Aldrich, P8833).

    Techniques: Migration, Western Blot, Expressing, Fluorescence, Single Cell Tracking, Transfection

    Expression of dominant-negative Rac1 blocks the induction of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N) or both constructs, were generated using the pRetroX-Tight-Pur Tet-On system

    Journal: Molecular cancer research : MCR

    Article Title: Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

    doi: 10.1158/1541-7786.MCR-10-0090

    Figure Lengend Snippet: Expression of dominant-negative Rac1 blocks the induction of vacuolization by active H-Ras. U251 cell lines capable of conditional expression of myc-H-Ras(G12V), FLAG-Rac1(T17N) or both constructs, were generated using the pRetroX-Tight-Pur Tet-On system

    Article Snippet: These were designated as U251 Tet-On. cDNAs encoding myc-H-Ras(G12V) and FLAG-Rac1(T17N) were subcloned into the NotI/MluI sites of the expression vector, pRetroX-Tight-Pur (Clontech).

    Techniques: Expressing, Dominant Negative Mutation, Construct, Generated

    EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.

    Journal: Cell reports

    Article Title: Polycomb- and Methylation-Independent Roles of EZH2 as a Transcription Activator

    doi: 10.1016/j.celrep.2018.11.035

    Figure Lengend Snippet: EZH2 Directly Activates AR Gene Transcription (A) EZH2 protein occupies the AR gene promoter. EZH2 ChIP-seq was performed in LNCaP cells with an antibody targeting endogenous EZH2 (top). HA ChIP-seq was performed using an anti-HA antibody in LNCaP cells with ectopic HA-EZH2 overexpression. Two biological replicates are shown (center and bottom). (B) ChIP-qPCR showing EZH2 binding along the AR gene promoter. ChIP was performed in LNCaP cells using anti-EZH2 and IgG antibodies and then subjected to qPCR using primer pairs targeting ~60-bp sliding windows within −1 kb to +3 kb of the AR gene. The x axis indicates the central location of the PCR products relative to the AR TSS. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (C) Different regions (of 400 bp) of the AR promoter (from 0 to +3 kb) were cloned into the pRetroX-Tight-Pur-Luc vector and transfected into 293T cells, which were then subjected to ChIP by anti-EZH2 or IgG. EZH2 occupancy at the ectopically expressed AR promoter was determined by qPCR using a common forward primer targeting the vector sequence and a reverse primer specific to each fragment. Data shown are mean (±SEM) of technical replicates from one representative experiment of two. (D) Various AR promoter regions were cloned into the pGL4.10 vector and transfected into 293T cells with either control pLVX or HA-EZH2 overexpression. Cells were then subjected to luciferase reporter assays. Results were normalized to the Renilla internal control. Data shown are mean (±SEM) of technical replicates from one representative experiment of three. (E) Schematic view of the AR promoter sequence starting from the transcription start site (TSS). The sgRNAs were labeled sgAR1 to 4, their sequences are shown in green font, and their distances to the AR TSS are marked as numbers. The primers (F2 and R2) for PCR validation are shown in purple. (F and G) The distal AR promoter region is required for EZH2 activation of AR transcription. LNCaP cells were infected with lentiCRISPR-Cas9 containing the pLENTI.V2 control, sgAR1+2, sgAR3+4, or sgAR1+4 for 48 hr. CRISPR-Cas9-mediated genome editing was confirmed by Sanger sequencing (F) and genomic DNA PCR (G) using primers F2 and R2 (indicated in A and E). (H) CRISPR-Cas9-edited LNCaP cells were transfected with control or EZH2-targeting siRNA for 48 hr. Total RNA was harvested and subjected to RT-PCR analysis using F2 and R2, which are expected to yield a wild-type (AR WT, top band with black asterisk) and a CRISPR-Cas9-deleted (AR del, bottom bands with red asterisk) AR mRNA.

    Article Snippet: The AR promoter P1, P2 and full length were inserted into pGL4.10 vector (catalog number E6651; Promega) by using XhoI and HindIII sites and AR fragments were cloned into the pRetroX-Tight-Pur-Luc plasmid (Clontech laboratories, Inc.) by using BamHI and BglII.

    Techniques: Chromatin Immunoprecipitation, Over Expression, Real-time Polymerase Chain Reaction, Binding Assay, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transfection, Sequencing, Luciferase, Labeling, Activation Assay, Infection, CRISPR, Reverse Transcription Polymerase Chain Reaction