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    Thermo Fisher gene exp dyx1c1 ccpg1 hs00370049 m1
    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of <t>DYX1C1</t> and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.
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    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR

    The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Functional Assay, Sequencing, Luciferase, Expressing, Plasmid Preparation, Transfection, Construct

    Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Marker, Quantitative RT-PCR

    Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Staining, Marker