pcr amplification  (Millipore)


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    PCR amplification plate
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    axypcr96hsc
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    Structured Review

    Millipore pcr amplification
    Long range ribosomal <t>PCR</t> Amplifications of the 3.5 kb target from Fall specimens with Dream Taq ™. M: <t>DNA</t> markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.

    https://www.bioz.com/result/pcr amplification/product/Millipore
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    pcr amplification - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease"

    Article Title: Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease

    Journal: Journal of Nematology

    doi: 10.21307/jofnem-2020-016

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with Dream Taq ™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with Dream Taq ™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of TaKaRa Ex Taq ® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq ® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq ® system and Dream Taq ™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.
    Figure Legend Snippet: PCR performance of TaKaRa Ex Taq ® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq ® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq ® system and Dream Taq ™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: Dream Taq ™; 5, 6, 7 and 8: Pfu ; 9, 10, 11and 12: Dream Taq ™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: Dream Taq ™; 5, 6, 7 and 8: Pfu ; 9, 10, 11and 12: Dream Taq ™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both Dream Taq ™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: Dream Taq ™ PCR buffer; B: Pfu PCR buffer.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both Dream Taq ™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: Dream Taq ™ PCR buffer; B: Pfu PCR buffer.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq ® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: Dream Taq ™; B: 18 S locus (1.7 kb) by Dream Taq ™, C: ITS and 28 S loci (1.9 kb) by Dream Taq ™; D: TaKaRa Ex Taq ® system.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq ® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: Dream Taq ™; B: 18 S locus (1.7 kb) by Dream Taq ™, C: ITS and 28 S loci (1.9 kb) by Dream Taq ™; D: TaKaRa Ex Taq ® system.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: Dream Taq ™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: Dream Taq ™ and Pfu ; 4, 5 and 6: Dream Taq ™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and Dream Taq ™ in 0.625 units.
    Figure Legend Snippet: PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: Dream Taq ™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: Dream Taq ™ and Pfu ; 4, 5 and 6: Dream Taq ™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and Dream Taq ™ in 0.625 units.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Concentration Assay

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by Dream Taq ™, B: ITS and 28 S loci (1.9 kb) by Dream Taq ™; C: Dream Taq ™ and PicoMaxx™ High Fidelity PCR System combined.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by Dream Taq ™, B: ITS and 28 S loci (1.9 kb) by Dream Taq ™; C: Dream Taq ™ and PicoMaxx™ High Fidelity PCR System combined.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of Taq 2000™, Platinum™ Taq and Dream Taq ™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq 2000™; 4, 5, 6 and NC by Platinum™ Taq ; 7, 8, 9 and NC by Dream Taq ™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq 2000™ or Dream Taq ™ in each reaction was aligned with Platinum™ Taq in 1.25 units.
    Figure Legend Snippet: PCR performance of Taq 2000™, Platinum™ Taq and Dream Taq ™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq 2000™; 4, 5, 6 and NC by Platinum™ Taq ; 7, 8, 9 and NC by Dream Taq ™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq 2000™ or Dream Taq ™ in each reaction was aligned with Platinum™ Taq in 1.25 units.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Concentration Assay

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.
    Figure Legend Snippet: PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    2) Product Images from "Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis"

    Article Title: Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-29-139

    Effects of TGF-β1 on expression of collagen III and fibronectin mRNA in HPMCs . Serum-starved HPMCs were incubated with TGF-β1 (2 or 10 ng/ml) for up to 72 h and RNA was then isolated and subjected to semi-quantitative RT-PCR analysis of collagen III (A) and fibronectin (B). Expression of β-actin was used as a loading control.
    Figure Legend Snippet: Effects of TGF-β1 on expression of collagen III and fibronectin mRNA in HPMCs . Serum-starved HPMCs were incubated with TGF-β1 (2 or 10 ng/ml) for up to 72 h and RNA was then isolated and subjected to semi-quantitative RT-PCR analysis of collagen III (A) and fibronectin (B). Expression of β-actin was used as a loading control.

    Techniques Used: Expressing, Incubation, Isolation, Quantitative RT-PCR

    3) Product Images from "Interleukin-8/CXCL8 is a growth factor for human lung cancer cells"

    Article Title: Interleukin-8/CXCL8 is a growth factor for human lung cancer cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6602227

    Expression of IL-8 mRNA and protein in lung cancer cell lines. Expression of IL-8 mRNA was measured by RT–PCR ( A ). A 1 μ g portion of total RNA was reverse-transcribed for PCR reactions of IL-8 and control GAPDH. The expected 289 bp band of IL-8 mRNA was strongly expressed in A549, H460 and MOR/P, but was undetectable in all SCLC cell lines. Production of IL-8 protein was measured by ELISA ( B ). Conditioned medium was collected after 1 × 10 6 cells were cultured in serum-free RPMI medium for 48 h. Each bar is the mean±s.e. of three determinations from two independent experiments.
    Figure Legend Snippet: Expression of IL-8 mRNA and protein in lung cancer cell lines. Expression of IL-8 mRNA was measured by RT–PCR ( A ). A 1 μ g portion of total RNA was reverse-transcribed for PCR reactions of IL-8 and control GAPDH. The expected 289 bp band of IL-8 mRNA was strongly expressed in A549, H460 and MOR/P, but was undetectable in all SCLC cell lines. Production of IL-8 protein was measured by ELISA ( B ). Conditioned medium was collected after 1 × 10 6 cells were cultured in serum-free RPMI medium for 48 h. Each bar is the mean±s.e. of three determinations from two independent experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

    Expression of CXCR1 and CXCR2 in lung cancer cell lines. Expression of CXCR1 and CXCR2 proteins on the cell surface was measured by flow cytometry with mouse anti-human CXCR1 (R1) and CXCR2 (R2) and mouse IgG as control (C). Representative flow cytometric histograms of A549 and Lu165 showing the low expressions of CXCR1 and CXCR2 in A549 ( A ) and high expressions of CXCR1 and CXCR2 in Lu165 ( B ), respectively. Percentage of positive cells of CXCR1 and CXCR2 in nine lung cancer cell lines are summarised in ( C ). Each bar is the mean±s.e. of four independent experiments. Expression of CXCR1 and CXCR2 mRNA was measured by RT–PCR in A549 and H460 (as a positive control) ( D ). Total RNA (1.5 μ g) was reverse-transcribed for PCR reactions of CXCR1, CXCR2 and control GAPDH. The expected 512 bp band of CXCR1 was expressed in A549 and H460. The expected 202 bp band of CXCR2 was expressed in control cell H460 but not in A549 cells.
    Figure Legend Snippet: Expression of CXCR1 and CXCR2 in lung cancer cell lines. Expression of CXCR1 and CXCR2 proteins on the cell surface was measured by flow cytometry with mouse anti-human CXCR1 (R1) and CXCR2 (R2) and mouse IgG as control (C). Representative flow cytometric histograms of A549 and Lu165 showing the low expressions of CXCR1 and CXCR2 in A549 ( A ) and high expressions of CXCR1 and CXCR2 in Lu165 ( B ), respectively. Percentage of positive cells of CXCR1 and CXCR2 in nine lung cancer cell lines are summarised in ( C ). Each bar is the mean±s.e. of four independent experiments. Expression of CXCR1 and CXCR2 mRNA was measured by RT–PCR in A549 and H460 (as a positive control) ( D ). Total RNA (1.5 μ g) was reverse-transcribed for PCR reactions of CXCR1, CXCR2 and control GAPDH. The expected 512 bp band of CXCR1 was expressed in A549 and H460. The expected 202 bp band of CXCR2 was expressed in control cell H460 but not in A549 cells.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction

    4) Product Images from "Mechanistically Distinct Mouse Models for CRX-Associated Retinopathy"

    Article Title: Mechanistically Distinct Mouse Models for CRX-Associated Retinopathy

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004111

    Differential expression of mutant CRX protein/RNA in K-IN mouse retinas. A–G . Paraffin embedded sagittal sections of P10 mouse retinas were stained with the mouse monoclonal CRX M02 antibody (Abnova) and imaged by fluorescent microscopy. ONL-outer nuclear layer, INL-inner nuclear layer, GCL-ganglion cell layer. H . SDS-PAGE and Western blot analyses of CRX proteins made by the indicated mouse strains at P10, using the rabbit polyclonal CRX 119b-1 (α-CRX) antibody [7] and mouse monoclonal anti-β-ACTIN (α-BACT, Sigma-Aldrich). Positive bands correlating with the ∼37 kD full-length CRX and ∼27 kD truncated CRX [E168d2] are visible. Lanes are numbered for reference (below). I . CRX protein levels were quantified by measuring the intensities of the CRX [E168d2] and full-length bands normalized to the β-ACTIN control using LI-COR Odyssey Image Studio software. The results are presented as fold changes (FC) relative to full-length CRX level in WT retina. (*p≤0.05) J . Crx mRNA levels were determined by quantitative real-time PCR using allele specific PCR primer pairs. Separate primer pairs were used to amplify WT Crx alone and total Crx ( WT + mutant ) in E168d2 and R90W mice (see Materials and Methods ). The results are presented as FC relative to WT retina. (*p≤0.05).
    Figure Legend Snippet: Differential expression of mutant CRX protein/RNA in K-IN mouse retinas. A–G . Paraffin embedded sagittal sections of P10 mouse retinas were stained with the mouse monoclonal CRX M02 antibody (Abnova) and imaged by fluorescent microscopy. ONL-outer nuclear layer, INL-inner nuclear layer, GCL-ganglion cell layer. H . SDS-PAGE and Western blot analyses of CRX proteins made by the indicated mouse strains at P10, using the rabbit polyclonal CRX 119b-1 (α-CRX) antibody [7] and mouse monoclonal anti-β-ACTIN (α-BACT, Sigma-Aldrich). Positive bands correlating with the ∼37 kD full-length CRX and ∼27 kD truncated CRX [E168d2] are visible. Lanes are numbered for reference (below). I . CRX protein levels were quantified by measuring the intensities of the CRX [E168d2] and full-length bands normalized to the β-ACTIN control using LI-COR Odyssey Image Studio software. The results are presented as fold changes (FC) relative to full-length CRX level in WT retina. (*p≤0.05) J . Crx mRNA levels were determined by quantitative real-time PCR using allele specific PCR primer pairs. Separate primer pairs were used to amplify WT Crx alone and total Crx ( WT + mutant ) in E168d2 and R90W mice (see Materials and Methods ). The results are presented as FC relative to WT retina. (*p≤0.05).

    Techniques Used: Expressing, Mutagenesis, Staining, Microscopy, SDS Page, Western Blot, Software, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Mouse Assay

    5) Product Images from "A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division"

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

    Journal: eLife

    doi: 10.7554/eLife.10767

    Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004
    Figure Legend Snippet: Cell cycle progression and complementation of cdkg1-2 . ( A ) Schematic representation of the CDKG1 locus and cdkg1-2 allele caused by insertion of a NIT1 plasmid and accompanying deletion (upper bracketed region). Coordinates are from genome assembly V5.5 taken from Phytozome ( http://phytozome.jgi.doe.gov ). Tall black rectangles represent exons and medium rectangles represent untranslated regions (UTR). Thin lines represent introns and intergenic regions. The lower bracketed region marks the 1.7kb 3’ UTR of CDKG1. Arrows represent the transcriptional starts and direction of transcription for URH1 and CDKG1 . Arrowheads mark binding sites for PCR primers used for genotyping: a1/b1 for CDKG1 last exon and adjacent 3’ UTR region, a4/b1 for NIT1 and CDKG1 3’ UTR junction (see Supplementary file 2 for detailed information). The 7 kb genomic region of the CDKG1 locus that was used to generate the HA-gCDKG1 complementation construct is shown below. The orange rectangle indicates the 3xHA tag located just downstream of the start codon. Primer sets a2/b2 and a3/b2 were used in RT-PCR experiments to amplify only HA-CDKG1 (a2/b2) or both endogenous and HA-CDKG1 cDNAs (a3/b2). ( B ) Ethidium bromide stained agarose gel showing genotyping PCR results for indicated strains. wt , wild-type; cdkg1-2, CDKG1 mutant strain; wt:HA-gCDKG1, transgenic line expressing HA-gCDKG1 ;. Results from representative non-complemented (1) and complemented (2, 3) progeny from a cross between cdkg1-2 and wt::HA-gCDKG1 are shown. Gene specific primer sets are shown on the right. Size phenotypes (large or wild type (WT)) are shown below. * Primer dimer. ( C ) Ethidium bromide stained agarose gel of RT-PCR products showing expression of HA-CDKG1 in a complemented cdkg1-2::HA-gCDKG1 strain. Strain genotypes are as described in panel ( B ). Gene specific amplicons and primer sets described in panel ( A ) are indicated to the right of each gel image. GBLP is an internal control. ( D ) Quantitative RT-PCR showing CDKG1 mRNA levels in synchronized wild-type ( WT ) or cdkg1-2::HA-gCDKG1 strains. All data were normalized to expression of internal control gene GBLP . Relative expression levels are shown based on values at time 0 hr that were set to 1. Error bars: S.D. of three replicates. ( E ) 12 hr light/12 hr dark synchronized wild type (black lines) and cdkg1-2 (orange lines) strains were monitored for passage through commitment (dashed lines), and for mitotic index (percentage in S/M phases) by light microscopy with fixed samples. The median cell size and time when the cultures had ~60% Committed cells is indicated by the dashed lines. DOI: http://dx.doi.org/10.7554/eLife.10767.004

    Techniques Used: Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Construct, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Mutagenesis, Transgenic Assay, Expressing, Quantitative RT-PCR, Light Microscopy

    Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009
    Figure Legend Snippet: Expression profiles and interactions between D cyclins, CDKG1 and MAT3/RBR during the cell cycle. ( A ) Ethidium bromide stained agarose gels showing amplification of cDNAs made with RNA samples taken from synchronized cultures in a 14 hr light/10 hr dark cycle at four stages: daughter cells (1 hr light), post-commitment cells (10 hr light), S/M phase cells (1 hr dark) and post-mitotic cells (4 hr dark). Primers used are listed in Supplementary file 2 and the number of PCR amplification cycles used for each reaction is displayed on the right. GBLP is an internal control. ( B ) Profiles of CDKG1, CDKB1 and GBLP mRNAs as described in ( A ), except the pre-commitment sample was from 4 hr light. ( C ) Interaction of CYCD3 AxAxA mutant with CDKG1 and MAT3. Mutated CYCD3 (LxCxE→AxAxA) was fused to the Gal4 activation domain (AD) and tested in an Y2H assay with MAT3/RBR and CDKG1. Empty indicates vector-only with no fusion protein. Growth of two independent co-transformants is shown, and the relative strength of interaction is indicated by -, no interaction, +, weak interaction, and +++, very strong interaction. ( D ) Bar graph shows relative kinase activity of IVT CDKG1, CDKG1 kd and CDKG1+CYCD3 using GST-MAT3 as a substrate. The amount of 32 P-labeled GST-MAT3 from each reaction was normalized to the value from lane 2. Data are expressed as the mean of three independent experiments. Error bar: S.D. ( E ) Immunoprecipitation (IP) from whole cell extracts, fractionation by SDS-PAGE and detection of CDKG1 by Western blotting using polyclonal antisera raised against full length CDGK1. All strains used were synchronized in S/M phase. Lanes 1,2 are concentrated input fractions prior to IP from indicated strains. Anti-HA (lanes 3,4) or anti-CDKG1 (lanes 5–8) antibodies were used for IPs as indicated and the supernatant (lanes 3,5,7) or pellet (lanes 4,6,8) fractions probed using anti-CDKG1. The anti-CDKG1 antibody detects a single band near the predicted molecular weight of 44 kDa that is not present in cdkg1-2 mutants. ( F ) Schematic of the regulatory hierarchy for the Chlamydomonas mitotic sizer pathway (left side) and the metazoan G1 control pathway (right side). Both pathways integrate internal and/or external signals for cell cycle progression through D-cyclin dependent CDKs that phosphorylate RB-related proteins. DOI: http://dx.doi.org/10.7554/eLife.10767.009

    Techniques Used: Expressing, Staining, Amplification, Polymerase Chain Reaction, Mutagenesis, Activation Assay, Y2H Assay, Plasmid Preparation, Activity Assay, Labeling, Immunoprecipitation, Fractionation, SDS Page, Western Blot, Molecular Weight

    6) Product Images from "Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens"

    Article Title: Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002938

    Phosphorylated status of ChvI regulates T6SS expression and Hcp secretion. A. tumefaciens wild-type C58 strain containing the empty vector (V) or one of the plasmids expressing ChvG, ChvI, ChvI(D52E) or ChvI(D52A) grown in AB-MES (pH 7.0 or 5.5) at 25°C for 6 h was analyzed for T6SS expression and Hcp secretion. ( A ) Total proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. ( B ) qRT-PCR analysis of mRNA levels of fha1 , atu4343 , hcp , and atu4349 . Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates. ( C ) Total (T) and secreted (S) proteins were analyzed by western blot analysis with antibodies against Hcp and RpoA. ( D ) Total and secreted (Sup) proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, Hcp, and RpoA. RpoA was an internal control. The positions of molecular mass markers (in kDa) are indicated on the left.
    Figure Legend Snippet: Phosphorylated status of ChvI regulates T6SS expression and Hcp secretion. A. tumefaciens wild-type C58 strain containing the empty vector (V) or one of the plasmids expressing ChvG, ChvI, ChvI(D52E) or ChvI(D52A) grown in AB-MES (pH 7.0 or 5.5) at 25°C for 6 h was analyzed for T6SS expression and Hcp secretion. ( A ) Total proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. ( B ) qRT-PCR analysis of mRNA levels of fha1 , atu4343 , hcp , and atu4349 . Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates. ( C ) Total (T) and secreted (S) proteins were analyzed by western blot analysis with antibodies against Hcp and RpoA. ( D ) Total and secreted (Sup) proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, Hcp, and RpoA. RpoA was an internal control. The positions of molecular mass markers (in kDa) are indicated on the left.

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    Effect of acidity on expression and protein stability of ChvG and ExoR. ( A ) qRT-PCR analysis of mRNA levels of chvG , chvI , and exoR for wild-type C58 strain grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for 6 h. Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates. ( B ) Total proteins isolated from ΔchvG ΔexoR expressing pChvG-HA and pExoR grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for the indicated times underwent western blot analysis with antibodies against ExoR (filled arrow), HA (for detecting ChvG-HA), and RpoA. The protein level of ChvG-HA was quantified and normalized to the level of endogenous RpoA. The amount of ChvG-HA at 0 h was set to 1. Data are mean ± SD of 2 biological replicates. ( C ) Equal volumes of total proteins (T), periplasmic fraction (P), and spheroplast pellets (S) isolated from ΔexoR (pExoR) grown in AB-MES (pH 7.0) at 25°C for 6 h were analyzed by western blot analysis with antibodies against ExoR, ActC, and RpoA. ActC was a control of periplasmic protein. ( D ) Total proteins isolated from ΔchvG ΔexoR containing the empty vector (V) or one of the plasmids expressing pChvG-HA and pExoR grown in AB-MES (pH 7.0) or AB-MES (pH 5.5) in the presence of chloramphenicol at 25°C for the indicated times underwent western blot analysis with antibodies against ExoR (filled arrow), HA (for detecting ChvG-HA), and RpoA. The protein level of precursor ExoR (ExoRp) and mature ExoR (ExoRm), and ChvG-HA were quantified with the level present at 0 h set to 100%. Data are mean ± SD of 3 biological replicates. ( E ) Total proteins isolated from ΔchvG expressing ChvG-HA grown in AB-MES (pH 7.0 or pH 5.5) in the presence of chloramphenicol at 25°C for the indicated times underwent western blot analysis with antibody HA (for detecting ChvG-HA). The protein level of ChvG-HA was quantified with the level present at 0 h was set to 100%. Data are mean ± SD of 2 biological replicates.
    Figure Legend Snippet: Effect of acidity on expression and protein stability of ChvG and ExoR. ( A ) qRT-PCR analysis of mRNA levels of chvG , chvI , and exoR for wild-type C58 strain grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for 6 h. Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates. ( B ) Total proteins isolated from ΔchvG ΔexoR expressing pChvG-HA and pExoR grown in AB-MES (pH 7.0 or pH 5.5) at 25°C for the indicated times underwent western blot analysis with antibodies against ExoR (filled arrow), HA (for detecting ChvG-HA), and RpoA. The protein level of ChvG-HA was quantified and normalized to the level of endogenous RpoA. The amount of ChvG-HA at 0 h was set to 1. Data are mean ± SD of 2 biological replicates. ( C ) Equal volumes of total proteins (T), periplasmic fraction (P), and spheroplast pellets (S) isolated from ΔexoR (pExoR) grown in AB-MES (pH 7.0) at 25°C for 6 h were analyzed by western blot analysis with antibodies against ExoR, ActC, and RpoA. ActC was a control of periplasmic protein. ( D ) Total proteins isolated from ΔchvG ΔexoR containing the empty vector (V) or one of the plasmids expressing pChvG-HA and pExoR grown in AB-MES (pH 7.0) or AB-MES (pH 5.5) in the presence of chloramphenicol at 25°C for the indicated times underwent western blot analysis with antibodies against ExoR (filled arrow), HA (for detecting ChvG-HA), and RpoA. The protein level of precursor ExoR (ExoRp) and mature ExoR (ExoRm), and ChvG-HA were quantified with the level present at 0 h set to 100%. Data are mean ± SD of 3 biological replicates. ( E ) Total proteins isolated from ΔchvG expressing ChvG-HA grown in AB-MES (pH 7.0 or pH 5.5) in the presence of chloramphenicol at 25°C for the indicated times underwent western blot analysis with antibody HA (for detecting ChvG-HA). The protein level of ChvG-HA was quantified with the level present at 0 h was set to 100%. Data are mean ± SD of 2 biological replicates.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Western Blot, Plasmid Preparation

    T6SS expression and Hcp secretion are positively regulated by ChvG/ChvI and negatively regulated by ExoR. A. tumefaciens wild-type strain C58, ΔchvG , ΔchvI , ΔexoR , and ΔchvG ΔexoR mutant strains grown in AB-MES (pH 7.0) at 25°C for 6 h were analyzed for T6SS expression and secretion. ( A ) Total proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. ( B ) Total (T) and secreted (S) proteins were analyzed by western blot analysis with antibodies against Hcp and RpoA. The non-secreted protein RpoA was an internal control. The positions of molecular mass markers (in kDa) are indicated on the left. ( C ) qRT-PCR analysis of mRNA levels of indicated genes. Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates.
    Figure Legend Snippet: T6SS expression and Hcp secretion are positively regulated by ChvG/ChvI and negatively regulated by ExoR. A. tumefaciens wild-type strain C58, ΔchvG , ΔchvI , ΔexoR , and ΔchvG ΔexoR mutant strains grown in AB-MES (pH 7.0) at 25°C for 6 h were analyzed for T6SS expression and secretion. ( A ) Total proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. ( B ) Total (T) and secreted (S) proteins were analyzed by western blot analysis with antibodies against Hcp and RpoA. The non-secreted protein RpoA was an internal control. The positions of molecular mass markers (in kDa) are indicated on the left. ( C ) qRT-PCR analysis of mRNA levels of indicated genes. Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates.

    Techniques Used: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR

    7) Product Images from "Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells"

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw312

    Effect of Stau1 on HCV replication and translation. ( A ) Downregulation of Stau1 inhibits HCV replication and translation. Huh7.5 cells transfected with Stau1 siRNA or control siRNA were grown for 24 h, then infected with JFH1 HCV. Forty-eight hours later, cell lysates were prepared, and western blotted for Stau1, NS5A, NS5B and RT-PCR on total RNA to determine levels of HCV RNA and GAPDH mRNA. Lane 1, untransfected controls; Lane 2, cells transfected with control siRNA; Lane 3, cells transfected with Stau1 siRNA. The middle panel shows quantitation of WB and RT-PCR bands. The right panel shows quantitative RT PCR of HCV RNA and GAPDH mRNA. Relative fold change in HCV RNA was calculated by normalizing the amount of GAPDH mRNA in each sample. All experiments were done in triplicate for each data point. ( B ) Overexpression of Stau1 enhances HCV replication and translation. Huh7.5 cells transfected with overexpressing Stau1 clone ( lane 3 ) or vector alone ( lane 2 ) were grown for 24 h, then infected with JFH1 HCV. Forty-eight hours later, cells were analyzed for the expression of Stau1, NS5A, NS5B and actin by WB. Total RNA was isolated from another set of experiments and analyzed for HCV RNA and GAPDH mRNA by RT-PCR. Lane 1, control; Lane 2, vector control; Lane 3, Stau1 overexpressed. The middle panel shows the quantitation of WB and RT-PCR bands. The right panel shows quantitative RT PCR of HCV RNA. ( C ) Effect of exogenously added Stau1 on in-vitro endogenous HCV replication in cell-free replication lysate. Endogenous HCV replication of cell-free replication lysate was done as described in the Materials and Methods ( 41 , 70 ). Lanes 1 and 2, control replication lysate alone incubated at 0 and 3 h, respectively. Lane 3–6 cell-free replication lysate supplemented with increasing concentration (0.25–1.5 pmol) of either Stau1 (top panel) or Auf1 (middle panel).
    Figure Legend Snippet: Effect of Stau1 on HCV replication and translation. ( A ) Downregulation of Stau1 inhibits HCV replication and translation. Huh7.5 cells transfected with Stau1 siRNA or control siRNA were grown for 24 h, then infected with JFH1 HCV. Forty-eight hours later, cell lysates were prepared, and western blotted for Stau1, NS5A, NS5B and RT-PCR on total RNA to determine levels of HCV RNA and GAPDH mRNA. Lane 1, untransfected controls; Lane 2, cells transfected with control siRNA; Lane 3, cells transfected with Stau1 siRNA. The middle panel shows quantitation of WB and RT-PCR bands. The right panel shows quantitative RT PCR of HCV RNA and GAPDH mRNA. Relative fold change in HCV RNA was calculated by normalizing the amount of GAPDH mRNA in each sample. All experiments were done in triplicate for each data point. ( B ) Overexpression of Stau1 enhances HCV replication and translation. Huh7.5 cells transfected with overexpressing Stau1 clone ( lane 3 ) or vector alone ( lane 2 ) were grown for 24 h, then infected with JFH1 HCV. Forty-eight hours later, cells were analyzed for the expression of Stau1, NS5A, NS5B and actin by WB. Total RNA was isolated from another set of experiments and analyzed for HCV RNA and GAPDH mRNA by RT-PCR. Lane 1, control; Lane 2, vector control; Lane 3, Stau1 overexpressed. The middle panel shows the quantitation of WB and RT-PCR bands. The right panel shows quantitative RT PCR of HCV RNA. ( C ) Effect of exogenously added Stau1 on in-vitro endogenous HCV replication in cell-free replication lysate. Endogenous HCV replication of cell-free replication lysate was done as described in the Materials and Methods ( 41 , 70 ). Lanes 1 and 2, control replication lysate alone incubated at 0 and 3 h, respectively. Lane 3–6 cell-free replication lysate supplemented with increasing concentration (0.25–1.5 pmol) of either Stau1 (top panel) or Auf1 (middle panel).

    Techniques Used: Transfection, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Expressing, Isolation, In Vitro, Incubation, Concentration Assay

    8) Product Images from "Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres"

    Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres

    Journal: Genome Biology

    doi: 10.1186/gb-2007-8-7-r148

    The BBB neocentromere contains a major and a minor centromere chromatin domain. DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-A, CENP-C, and CENP-H from cell line BBB was hybridized to a custom made microarray containing 257 unique polymerase chain reaction (PCR) fragments. Three independent biological replicates were performed for each antibody, and the scale normalized mean log 2 Cy-5:Cy-3 intensity ratios (ChIP to input), were plotted on the y-axis with the standard error (SE) for each PCR fragment. Intensity ratios at least three times the standard deviation (SD) from the background mean (dashed line) were considered positives (see Materials and methods). An alpha satellite containing plasmid was included as a positive control (far right). (a) Centromere protein (CENP)-A ChIP. The major CENP-A domain was about 80.3 kilobases (kb; shaded region), with positive intensity ratios 1.17 to 2.46. The minor domain was about 8.5 kb (shaded region) and was approximately 162 kb downstream from the major domain; intensity ratios were 1.14 to 1.33. Background experimental mean was -0.39 ± 0.47 SD, one-tailed distribution cut-off was ≤ 0.68, positive values were ≥ 1.02 (dashed line). Alpha satellite = 1.63 ± 0.18 SE. (b) CENP-C ChIP. Major CENP-C domain was 87.8 kb (shaded region). Intensity ratios were 0.67 to 3.41. Minor domain was 8.5 kb; intensity ratios were 0.65 to 1.07 (shaded region). Background experimental mean was -0.37 ± 0.34 SD, one-tailed distribution cut-off was ≤ 0.31, positive values were ≥ 0.65 (dashed line). Alpha satellite = 2.36 ± 0.70 SE. (c) CENP-H ChIP. Major CENP-H domain was about 86.3 kb (shaded region), and positive intensity ratios were 0.64 to 3.35. Minor domain was about 1.9 kb (shaded region), and intensity ratios were 0.82 and 1.14. Background experimental mean was -0.33 ± 0.32 SD, one-tailed distribution cutoff was ≤ 0.56, positive values were ≥ 0.63 (dashed lines). Alpha sat = 2.06 ± 0.59 SE. (d) The 2.3 megabase (Mb) region included in the PCR CHIP. The central 350 kb region, covered by PCR fragments at high density. The adjacent megabase on either side of the central region, shown at a 10 fold reduced scale, was covered by PCR fragments at decreasing density. PCR microarray fragments listed in Table 1, found at the edges of CENP-A, CENP-C and CENP-H domains, and the negative values within the first domain, are shown. The major and minor chromatin domains are shown by the rectangles. The tiling path of the unique sequenced regions of each bacterial artificial chromosome (BAC) and their overlaps are shown within the 350 kb region. The corresponding Repeat Masker data from the Human Genome Browser at UCSC and thegenes in the area are indicated [50].
    Figure Legend Snippet: The BBB neocentromere contains a major and a minor centromere chromatin domain. DNA obtained from chromatin immunoprecipitation (ChIP) using antibodies to CENP-A, CENP-C, and CENP-H from cell line BBB was hybridized to a custom made microarray containing 257 unique polymerase chain reaction (PCR) fragments. Three independent biological replicates were performed for each antibody, and the scale normalized mean log 2 Cy-5:Cy-3 intensity ratios (ChIP to input), were plotted on the y-axis with the standard error (SE) for each PCR fragment. Intensity ratios at least three times the standard deviation (SD) from the background mean (dashed line) were considered positives (see Materials and methods). An alpha satellite containing plasmid was included as a positive control (far right). (a) Centromere protein (CENP)-A ChIP. The major CENP-A domain was about 80.3 kilobases (kb; shaded region), with positive intensity ratios 1.17 to 2.46. The minor domain was about 8.5 kb (shaded region) and was approximately 162 kb downstream from the major domain; intensity ratios were 1.14 to 1.33. Background experimental mean was -0.39 ± 0.47 SD, one-tailed distribution cut-off was ≤ 0.68, positive values were ≥ 1.02 (dashed line). Alpha satellite = 1.63 ± 0.18 SE. (b) CENP-C ChIP. Major CENP-C domain was 87.8 kb (shaded region). Intensity ratios were 0.67 to 3.41. Minor domain was 8.5 kb; intensity ratios were 0.65 to 1.07 (shaded region). Background experimental mean was -0.37 ± 0.34 SD, one-tailed distribution cut-off was ≤ 0.31, positive values were ≥ 0.65 (dashed line). Alpha satellite = 2.36 ± 0.70 SE. (c) CENP-H ChIP. Major CENP-H domain was about 86.3 kb (shaded region), and positive intensity ratios were 0.64 to 3.35. Minor domain was about 1.9 kb (shaded region), and intensity ratios were 0.82 and 1.14. Background experimental mean was -0.33 ± 0.32 SD, one-tailed distribution cutoff was ≤ 0.56, positive values were ≥ 0.63 (dashed lines). Alpha sat = 2.06 ± 0.59 SE. (d) The 2.3 megabase (Mb) region included in the PCR CHIP. The central 350 kb region, covered by PCR fragments at high density. The adjacent megabase on either side of the central region, shown at a 10 fold reduced scale, was covered by PCR fragments at decreasing density. PCR microarray fragments listed in Table 1, found at the edges of CENP-A, CENP-C and CENP-H domains, and the negative values within the first domain, are shown. The major and minor chromatin domains are shown by the rectangles. The tiling path of the unique sequenced regions of each bacterial artificial chromosome (BAC) and their overlaps are shown within the 350 kb region. The corresponding Repeat Masker data from the Human Genome Browser at UCSC and thegenes in the area are indicated [50].

    Techniques Used: Chromatin Immunoprecipitation, Microarray, Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Positive Control, One-tailed Test, BAC Assay

    qRT-PCR confirms two separate CenpA domains in the neocentromeric cell line BBB. (a) Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on equal amounts of total DNA obtained from centromere protein (CENP)-A chromatin immunoprecipitation (ChIP) DNA and Input DNA from BBB cell line. The thirty-four PCR primer pairs used (shown as black lines in the x-axis) amplified fragments from 150 to 250 base pairs contained within the 350 kb neocentromere region (see Figure 2). Each primer pair was assayed in at least three independent CENP-A ChIP experiments. The qRT-PCR results for each primer pair were expressed on the y-axis as the fold enhancement between the CENP-A ChIP DNA and input DNA (= 1.93 ΔCt(CENP-A-Input) ) normalized to the value obtained for the positive control alpha satellite DNA primer pair (far right). The shaded region indicates the area determined to be the CENP-A domain in Figure 2. (b) The 34 qRT-PCR primer pairs and the 133 PCR products from this region on the PCR microarray (Figure 2) are shown. qRT-PCR primers that amplified products wholly contained within a PCR microarray fragment are indicated by numbers in parentheses; the rest are labeled alphabetically. Only qRT-PCR fragments shown in Table 1 are indicated; information for all other primers can be found in the Additional data file 3. CENP-A domains derived from the PCR microarray data are indicated. Genome coordinates correspond to the region of chr13 from the Human Genome Browser at UCSC (hg17) [50].
    Figure Legend Snippet: qRT-PCR confirms two separate CenpA domains in the neocentromeric cell line BBB. (a) Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on equal amounts of total DNA obtained from centromere protein (CENP)-A chromatin immunoprecipitation (ChIP) DNA and Input DNA from BBB cell line. The thirty-four PCR primer pairs used (shown as black lines in the x-axis) amplified fragments from 150 to 250 base pairs contained within the 350 kb neocentromere region (see Figure 2). Each primer pair was assayed in at least three independent CENP-A ChIP experiments. The qRT-PCR results for each primer pair were expressed on the y-axis as the fold enhancement between the CENP-A ChIP DNA and input DNA (= 1.93 ΔCt(CENP-A-Input) ) normalized to the value obtained for the positive control alpha satellite DNA primer pair (far right). The shaded region indicates the area determined to be the CENP-A domain in Figure 2. (b) The 34 qRT-PCR primer pairs and the 133 PCR products from this region on the PCR microarray (Figure 2) are shown. qRT-PCR primers that amplified products wholly contained within a PCR microarray fragment are indicated by numbers in parentheses; the rest are labeled alphabetically. Only qRT-PCR fragments shown in Table 1 are indicated; information for all other primers can be found in the Additional data file 3. CENP-A domains derived from the PCR microarray data are indicated. Genome coordinates correspond to the region of chr13 from the Human Genome Browser at UCSC (hg17) [50].

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Positive Control, Microarray, Labeling, Derivative Assay

    CENP-A nucleosomes are interspersed at variable densities throughout the core centromeric domain. (a) The 87.8 kilobase (kb) major domain. Shown are the putative subdomains of centromere protein (CENP)-A, with higher densities indicated by darker shading. The polymerase chain reaction (PCR) microarray fragments are shown below (see Figure 2). The fragments examined using the oligo array are shown in gray. (b) DNA obtained from chromatin immunoprecipitation (ChIP) using CENP-A from cell line BBB was hybridized to a 70 mer oligonucleotide microarray containing two distinct subdomains of the major neocentromere domain, a 1.6 kb region at the 5' end (PCR fragments 3 and 4; Table 1) and a 2 kb region within the domain (PCR fragments 20 and 21). Three independent biological replicates were performed, and the mean log 2 Cy-5:Cy-3 intensity ratio (CENP-A ChIP to input) from each biologic replicate was scale normalized (SN). The result for each 70 mer oligomer is shown plotted on the y-axis with the standard error.
    Figure Legend Snippet: CENP-A nucleosomes are interspersed at variable densities throughout the core centromeric domain. (a) The 87.8 kilobase (kb) major domain. Shown are the putative subdomains of centromere protein (CENP)-A, with higher densities indicated by darker shading. The polymerase chain reaction (PCR) microarray fragments are shown below (see Figure 2). The fragments examined using the oligo array are shown in gray. (b) DNA obtained from chromatin immunoprecipitation (ChIP) using CENP-A from cell line BBB was hybridized to a 70 mer oligonucleotide microarray containing two distinct subdomains of the major neocentromere domain, a 1.6 kb region at the 5' end (PCR fragments 3 and 4; Table 1) and a 2 kb region within the domain (PCR fragments 20 and 21). Three independent biological replicates were performed, and the mean log 2 Cy-5:Cy-3 intensity ratio (CENP-A ChIP to input) from each biologic replicate was scale normalized (SN). The result for each 70 mer oligomer is shown plotted on the y-axis with the standard error.

    Techniques Used: Polymerase Chain Reaction, Microarray, Chromatin Immunoprecipitation

    9) Product Images from "The RECQL helicase prevents replication fork collapse during replication stress"

    Article Title: The RECQL helicase prevents replication fork collapse during replication stress

    Journal: Life Science Alliance

    doi: 10.26508/lsa.202000668

    RECQL prevents DNA double-strand break formation in human cancer cells. (A) RECQL expression levels of different cancer cell lines transfected with control and RECQL siRNAs measured by qRT-PCR. Error bars denote the SD of two independent experiments. (B) Tail moments of different cancer cell lines transfected with control and RECQL siRNAs. Mean of each condition is indicated by the red line and number. P -values are indicated above each cell line. (C, D) Tail moments of VU120T (C) and OCUB-M (D) cancer cells transfected with control and RECQL siRNAs and treated with and without 12.5 μM (C) and 6.25 μM (D) Mirin, respectively. SDs are plotted in black and red bars denote the mean. For (B, C, D), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
    Figure Legend Snippet: RECQL prevents DNA double-strand break formation in human cancer cells. (A) RECQL expression levels of different cancer cell lines transfected with control and RECQL siRNAs measured by qRT-PCR. Error bars denote the SD of two independent experiments. (B) Tail moments of different cancer cell lines transfected with control and RECQL siRNAs. Mean of each condition is indicated by the red line and number. P -values are indicated above each cell line. (C, D) Tail moments of VU120T (C) and OCUB-M (D) cancer cells transfected with control and RECQL siRNAs and treated with and without 12.5 μM (C) and 6.25 μM (D) Mirin, respectively. SDs are plotted in black and red bars denote the mean. For (B, C, D), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Software

    RECQL is essential for proliferation in replication stress conditions. (A) RECQL expression levels in TBP and TBP-RecqlKD MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (B) RECQL protein levels in TBP and TBP-RecqlKD MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP (black) and TBP-RecqlKD (grey) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Relative apoptosis of TBP (black) and TBP-RecqlKD (grey) MEFs cultured without FCS. Apoptosis was measured by fluorescent signal upon caspase 3 cleavage and normalized to cell confluency. For (C, D, E), error bars show SD. (F) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs cultured with FCS or for 7 d without FCS. For each condition, more than 50 cells were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test). (G) Immunofluorescence images of γH2AX and CldU foci in TBP-shRNA control and TBP-RecqlKD MEFs after 2 d of serum starvation. DNA was labelled with Topro3. In the merged picture, DNA is blue, γH2AX is green and CldU is red. Colocalization of yH2AX and CldU is seen as yellow foci. Scale bar = 12 μm. (H) Quantification of the number of γH2AX foci per nucleus in CldU-negative TBP-shRNA control and TBP-RecqlKD MEFs in the presence and absence of serum. Mean is indicated in red. Results of three independent experiments are pooled and significance is indicated ( t test).
    Figure Legend Snippet: RECQL is essential for proliferation in replication stress conditions. (A) RECQL expression levels in TBP and TBP-RecqlKD MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (B) RECQL protein levels in TBP and TBP-RecqlKD MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP (black) and TBP-RecqlKD (grey) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Relative apoptosis of TBP (black) and TBP-RecqlKD (grey) MEFs cultured without FCS. Apoptosis was measured by fluorescent signal upon caspase 3 cleavage and normalized to cell confluency. For (C, D, E), error bars show SD. (F) Tail moments of TBP (black) and TBP-RecqlKD (grey) MEFs cultured with FCS or for 7 d without FCS. For each condition, more than 50 cells were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test). (G) Immunofluorescence images of γH2AX and CldU foci in TBP-shRNA control and TBP-RecqlKD MEFs after 2 d of serum starvation. DNA was labelled with Topro3. In the merged picture, DNA is blue, γH2AX is green and CldU is red. Colocalization of yH2AX and CldU is seen as yellow foci. Scale bar = 12 μm. (H) Quantification of the number of γH2AX foci per nucleus in CldU-negative TBP-shRNA control and TBP-RecqlKD MEFs in the presence and absence of serum. Mean is indicated in red. Results of three independent experiments are pooled and significance is indicated ( t test).

    Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Software, Immunofluorescence, shRNA

    shRNA-based screen to identify genes essential for proliferation in replication stress conditions. (A) Schematic outline of the shRNA screen. TBP MEFs were infected with the lentiviral DNA damage response shRNA library and cultured in the absence or presence of FCS. ShRNA inserts were subsequently recovered by PCR and analyzed by next generation sequencing. ShRNAs that were depleted in the abscence of serum likely target genes essential for proliferation in replication stress conditions. (B) The shRNA screen identified expression of CHK1 and RECQL essential for proliferation in replication stress conditions. The x-axis shows the average number of sequencing reads at the start point. The y-axis depicts the fold change in abundance of shRNAs in the cells cultured without FCS versus with FCS. (C) CHK1 expression levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (D) CHK1 protein levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs. Anti-actin was used as loading control. (E) Growth curve of TBP MEFs with FCS (black) and without FCS (green), TBP-Chk1KD#1 MEFs with FCS (grey), and without FCS (blue) measured using the IncuCyte. Error bars show SD.
    Figure Legend Snippet: shRNA-based screen to identify genes essential for proliferation in replication stress conditions. (A) Schematic outline of the shRNA screen. TBP MEFs were infected with the lentiviral DNA damage response shRNA library and cultured in the absence or presence of FCS. ShRNA inserts were subsequently recovered by PCR and analyzed by next generation sequencing. ShRNAs that were depleted in the abscence of serum likely target genes essential for proliferation in replication stress conditions. (B) The shRNA screen identified expression of CHK1 and RECQL essential for proliferation in replication stress conditions. The x-axis shows the average number of sequencing reads at the start point. The y-axis depicts the fold change in abundance of shRNAs in the cells cultured without FCS versus with FCS. (C) CHK1 expression levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs measured by qRT-PCR. Error bars show SD of two independent experiments. (D) CHK1 protein levels in TBP, TBP-Chk1KD#1 and TBP-Chk1#2 MEFs. Anti-actin was used as loading control. (E) Growth curve of TBP MEFs with FCS (black) and without FCS (green), TBP-Chk1KD#1 MEFs with FCS (grey), and without FCS (blue) measured using the IncuCyte. Error bars show SD.

    Techniques Used: shRNA, Infection, Cell Culture, Polymerase Chain Reaction, Next-Generation Sequencing, Expressing, Sequencing, Quantitative RT-PCR

    Two independently generated TBP-RecqlKD MEF cell lines and cell cycle analysis of mitogen-deprived TBP-RecqlKD MEFs. (A) RECQL expression levels in TBP and TBP-RecqlKD MEFs generated by two newly designed shRNAs (TBP-RecqlKD#2 and TBP-RecqlKD#3) measured by qRT-PCR. Error bars show SD. (B) RECQL protein levels in TBP, TBP-RecqlKD#2 and TBP-RecqlKD#3 MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP-shRNA control (black), TBP-RecqlKD#2 (green) and TBP-RecqlKD#3 (blue) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Duration of G1 and G2 cell cycle phases of TBP and TBP-RecqlKD MEFs expressing FUCCI constructs (mKO-hCdt1 and mAG-hGEM) in the presence or absence of FCS. G1 phase: only mKO-hCdt1 expression. Early S phase: both mKO-hCdt1 and mAG-hGEM expression. S/G2 phase: only mAG-hGEM expression. Data from TBP MEFs were taken from Benedict et al (2020) . Error bars show SDs.
    Figure Legend Snippet: Two independently generated TBP-RecqlKD MEF cell lines and cell cycle analysis of mitogen-deprived TBP-RecqlKD MEFs. (A) RECQL expression levels in TBP and TBP-RecqlKD MEFs generated by two newly designed shRNAs (TBP-RecqlKD#2 and TBP-RecqlKD#3) measured by qRT-PCR. Error bars show SD. (B) RECQL protein levels in TBP, TBP-RecqlKD#2 and TBP-RecqlKD#3 MEFs. Anti-actin was used as loading control. (C, D) Growth curves of TBP-shRNA control (black), TBP-RecqlKD#2 (green) and TBP-RecqlKD#3 (blue) MEFs cultured in the presence (C) and absence of FCS (D) measured with the IncuCyte. (E) Duration of G1 and G2 cell cycle phases of TBP and TBP-RecqlKD MEFs expressing FUCCI constructs (mKO-hCdt1 and mAG-hGEM) in the presence or absence of FCS. G1 phase: only mKO-hCdt1 expression. Early S phase: both mKO-hCdt1 and mAG-hGEM expression. S/G2 phase: only mAG-hGEM expression. Data from TBP MEFs were taken from Benedict et al (2020) . Error bars show SDs.

    Techniques Used: Generated, Cell Cycle Assay, Expressing, Quantitative RT-PCR, shRNA, Cell Culture, Construct

    MRE11 dependence of double-strand break induction. (A, B) MRE11 expression levels in TBP (A) and TBP-RecqlKD (B) MEFs transfected with control (black) and MRE11 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (C) Tail moments of TBP and TBP-RecqlKD MEFs transfected with control (black) and MRE11 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (D) Tail moments of TBP MEFs untreated, immediately after 1 h 2 mM HU treatment or 30 min after washing away HU either in the absence or presence of 50 μM Mirin. (E, F) DNA2 expression levels in TBP-shRNA control (E) and TBP-RecqlKD (F) MEFs transfected with control (black) or DNA2 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (G) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and DNA2 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (H, I) MUS81 expression levels in TBP-shRNA control (H) and TBP-Recql KD (I) MEFs transfected with control (black) and MUS81 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (J) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and MUS81 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. For (C, D, G, J), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).
    Figure Legend Snippet: MRE11 dependence of double-strand break induction. (A, B) MRE11 expression levels in TBP (A) and TBP-RecqlKD (B) MEFs transfected with control (black) and MRE11 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (C) Tail moments of TBP and TBP-RecqlKD MEFs transfected with control (black) and MRE11 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (D) Tail moments of TBP MEFs untreated, immediately after 1 h 2 mM HU treatment or 30 min after washing away HU either in the absence or presence of 50 μM Mirin. (E, F) DNA2 expression levels in TBP-shRNA control (E) and TBP-RecqlKD (F) MEFs transfected with control (black) or DNA2 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (G) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and DNA2 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. (H, I) MUS81 expression levels in TBP-shRNA control (H) and TBP-Recql KD (I) MEFs transfected with control (black) and MUS81 (grey) siRNAs measured by qRT-PCR. Error bars show SDs. (J) Tail moments of TBP-shRNA control and TBP-RecqlKD MEFs transfected with control (black) and MUS81 (grey) siRNAs, measured immediately after 1 h 300 μM HU treatment. For (C, D, G, J), more than 50 cells for each condition were analyzed using CASP software. SDs are plotted in black and red bars denote the mean. Significance is indicated (one-way ANOVA nonparametric Kruskal–Wallis test).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, shRNA, Software

    10) Product Images from "RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris. RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris"

    Article Title: RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris. RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12938

    The effects of RpoN2 on binding to the promoter of Xanthomonas campestris pv. campestris fliC . (a) Relative expression of fliC as determined by RNA‐Seq. (b) Relative expression of fliC as determined by quantitative reverse transcription PCR. (c) and (d) Gel shift assay showing that RpoN2 and RpoN1 directly regulate fliC . RpoN2 and RpoN1 (0, 1, 2, 4, or 8 μM) were added to reaction mixtures containing 50 ng of probe DNA, and the reaction mixtures were separated on polyacrylamide gels. (e) The effect of RpoN on fliC gene expression was measured by assessing the β‐galactosidase activity of the fliC ‐ lacZ transcriptional fusions in the Xc1 wild‐type, Δ rpoN1 , Δ rpoN2 , and Δ rpoN1N2 strains. Error bars, means ± SD ( n = 3). * p
    Figure Legend Snippet: The effects of RpoN2 on binding to the promoter of Xanthomonas campestris pv. campestris fliC . (a) Relative expression of fliC as determined by RNA‐Seq. (b) Relative expression of fliC as determined by quantitative reverse transcription PCR. (c) and (d) Gel shift assay showing that RpoN2 and RpoN1 directly regulate fliC . RpoN2 and RpoN1 (0, 1, 2, 4, or 8 μM) were added to reaction mixtures containing 50 ng of probe DNA, and the reaction mixtures were separated on polyacrylamide gels. (e) The effect of RpoN on fliC gene expression was measured by assessing the β‐galactosidase activity of the fliC ‐ lacZ transcriptional fusions in the Xc1 wild‐type, Δ rpoN1 , Δ rpoN2 , and Δ rpoN1N2 strains. Error bars, means ± SD ( n = 3). * p

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Activity Assay

    11) Product Images from "The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins"

    Article Title: The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx703

    Raf is required for partitioning competence of Rep2 D22N and Rep2 AA . A cir 0 yeast strain was transformed with kanMX4 -tagged, amplification-incompetent ( flp − ) 2-μm plasmids (pKan-based) carrying a wild-type REP1 gene and the indicated version of the REP2 and RAF genes. Due to the absence of a functional FLP gene, plasmid missegregation events cannot be corrected by Flp-mediated copy number amplification, making efficient maintenance of the pKan-based plasmids dependent on Rep protein partitioning function. Transformants were cultured overnight (six to eight generations) in selective medium, and the fraction of plasmid-bearing cells determined by a plating assay. Results represent the average (±s.d.) of five independent transformants for each plasmid. Plasmid copy number in each culture was determined by polymerase chain reaction (PCR) using total DNA extracted from the transformants as template and quantifying the ratio of product obtained with primers specific for a plasmid relative to a chromosomal locus. This value was then corrected for the fraction of cells in the population containing plasmid to obtain the average plasmid copy number per plasmid-bearing cell (See Supplementary Figure S6 for details).
    Figure Legend Snippet: Raf is required for partitioning competence of Rep2 D22N and Rep2 AA . A cir 0 yeast strain was transformed with kanMX4 -tagged, amplification-incompetent ( flp − ) 2-μm plasmids (pKan-based) carrying a wild-type REP1 gene and the indicated version of the REP2 and RAF genes. Due to the absence of a functional FLP gene, plasmid missegregation events cannot be corrected by Flp-mediated copy number amplification, making efficient maintenance of the pKan-based plasmids dependent on Rep protein partitioning function. Transformants were cultured overnight (six to eight generations) in selective medium, and the fraction of plasmid-bearing cells determined by a plating assay. Results represent the average (±s.d.) of five independent transformants for each plasmid. Plasmid copy number in each culture was determined by polymerase chain reaction (PCR) using total DNA extracted from the transformants as template and quantifying the ratio of product obtained with primers specific for a plasmid relative to a chromosomal locus. This value was then corrected for the fraction of cells in the population containing plasmid to obtain the average plasmid copy number per plasmid-bearing cell (See Supplementary Figure S6 for details).

    Techniques Used: Transformation Assay, Amplification, Functional Assay, Plasmid Preparation, Cell Culture, Polymerase Chain Reaction

    Raf associates with STB and 2-μm plasmid gene promoters, and association with STB is dependent on Rep1. ( A ) A cir + yeast strain was transformed with an ARS/CEN plasmid expressing FLAG-tagged Raf under the control of a galactose-inducible promoter. Transformed yeast were cultured in medium containing galactose and chromatin was immunoprecipitated with antibodies specific for native Rep1 and Rep2, anti-FLAG (FLAG-Raf) and, as a negative control, anti-HA. The precipitated DNA was analyzed by semi-quantitative PCR with primers specific for the STB locus, for the divergent FLP/REP2 and REP1/RAF promoter regions ( FLP/REP2p and REP1/RAFp ), and, as a negative control, a chromosomal locus ( TRP1 ). The bar graph indicates ChIP efficiency as the percent of input DNA immunoprecipitated; results are average (±s.d.) from triplicate assays. Ethidium bromide-stained agarose gels of PCR products from a representative assay are shown below the graph. Template DNA amplified in ‘input’ PCR reactions is 10% of that amplified in ‘ChIP’ PCR reactions. Products obtained from neat and 1:4 dilutions of each template are shown. ( B ) A cir 0 yeast strain with STB integrated in the chromosome upstream of a HIS3 reporter gene was co-transformed with two galactose-inducible expression plasmids: one expressing B42 AD -HA (AD) or B42 AD -HA-Raf (AD-Raf), and the second, expressing either no protein (−), or expressing Rep1 or Rep2. Five-fold serial dilutions of co-transformants were spotted onto solid media that selected for the presence of the two plasmids, with galactose to induce Rep protein expression, and either containing histidine (+His), or lacking histidine (−His) and containing 3-aminotriazole (3-AT). Growth on the −His + 3-AT medium indicates recruitment of the AD fusion protein to STB .
    Figure Legend Snippet: Raf associates with STB and 2-μm plasmid gene promoters, and association with STB is dependent on Rep1. ( A ) A cir + yeast strain was transformed with an ARS/CEN plasmid expressing FLAG-tagged Raf under the control of a galactose-inducible promoter. Transformed yeast were cultured in medium containing galactose and chromatin was immunoprecipitated with antibodies specific for native Rep1 and Rep2, anti-FLAG (FLAG-Raf) and, as a negative control, anti-HA. The precipitated DNA was analyzed by semi-quantitative PCR with primers specific for the STB locus, for the divergent FLP/REP2 and REP1/RAF promoter regions ( FLP/REP2p and REP1/RAFp ), and, as a negative control, a chromosomal locus ( TRP1 ). The bar graph indicates ChIP efficiency as the percent of input DNA immunoprecipitated; results are average (±s.d.) from triplicate assays. Ethidium bromide-stained agarose gels of PCR products from a representative assay are shown below the graph. Template DNA amplified in ‘input’ PCR reactions is 10% of that amplified in ‘ChIP’ PCR reactions. Products obtained from neat and 1:4 dilutions of each template are shown. ( B ) A cir 0 yeast strain with STB integrated in the chromosome upstream of a HIS3 reporter gene was co-transformed with two galactose-inducible expression plasmids: one expressing B42 AD -HA (AD) or B42 AD -HA-Raf (AD-Raf), and the second, expressing either no protein (−), or expressing Rep1 or Rep2. Five-fold serial dilutions of co-transformants were spotted onto solid media that selected for the presence of the two plasmids, with galactose to induce Rep protein expression, and either containing histidine (+His), or lacking histidine (−His) and containing 3-aminotriazole (3-AT). Growth on the −His + 3-AT medium indicates recruitment of the AD fusion protein to STB .

    Techniques Used: Plasmid Preparation, Transformation Assay, Expressing, Cell Culture, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Staining, Polymerase Chain Reaction, Amplification

    12) Product Images from "Epstein-Barr Virus-Induced miR-155 Attenuates NF-?B Signaling and Stabilizes Latent Virus Persistence ▿Epstein-Barr Virus-Induced miR-155 Attenuates NF-?B Signaling and Stabilizes Latent Virus Persistence ▿ †"

    Article Title: Epstein-Barr Virus-Induced miR-155 Attenuates NF-?B Signaling and Stabilizes Latent Virus Persistence ▿Epstein-Barr Virus-Induced miR-155 Attenuates NF-?B Signaling and Stabilizes Latent Virus Persistence ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00752-08

    miR-155 stabilizes EBV genome copy number during latent infection. (A) EBV DNA copy number was assayed by real-time PCR of EBV dyad symmetry (DS) DNA relative to cellular actin in Raji cells transfected with miR-155 inhibitor or control. (B) Same as in panel A, except in LCL cells. (C) Raji cells were transfected with miR-155 inhibitor or control and then assayed for EBNA1 mRNA expression relative to actin using RT-PCR. (D) Same as in panel C, except in LCL cells. (E) Southern blot analysis of EBV genomic DNA (upper panel) and cellular telomere repeat DNA (Telo, lower panel) in control (Ctrl) or miR-155 inhibitor-transfected Raji cells. (F) Summary of genetic interactions of miR-155 relevant to EBV immortalization and stable latent infection. EBV LMP1 induces BIC/miR-155, which in turn attenuates NF-κB signaling and IKKɛ protein expression. miR-155 also contributes to EBV genome maintenance, in part by stimulating EBNA1 mRNA expression.
    Figure Legend Snippet: miR-155 stabilizes EBV genome copy number during latent infection. (A) EBV DNA copy number was assayed by real-time PCR of EBV dyad symmetry (DS) DNA relative to cellular actin in Raji cells transfected with miR-155 inhibitor or control. (B) Same as in panel A, except in LCL cells. (C) Raji cells were transfected with miR-155 inhibitor or control and then assayed for EBNA1 mRNA expression relative to actin using RT-PCR. (D) Same as in panel C, except in LCL cells. (E) Southern blot analysis of EBV genomic DNA (upper panel) and cellular telomere repeat DNA (Telo, lower panel) in control (Ctrl) or miR-155 inhibitor-transfected Raji cells. (F) Summary of genetic interactions of miR-155 relevant to EBV immortalization and stable latent infection. EBV LMP1 induces BIC/miR-155, which in turn attenuates NF-κB signaling and IKKɛ protein expression. miR-155 also contributes to EBV genome maintenance, in part by stimulating EBNA1 mRNA expression.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Southern Blot

    BIC RNA levels are highest in EBV-positive LCL cells. (A) BIC RNA levels were assayed by RT-PCR and normalized to actin RNA levels for various cell lines (DG75 is an EBV-negative BL cell line, Raji is EBV-positive BL cell line, LCL is an EBV-positive lymphoblastoid cell line, 293T is an EBV-negative HEK cell line, ZKO is a Zta knockout EBV bacmid-positive 293 cell line, and D98 is an EBV P3HR1 strain-positive HeLa cell line). (B and C) Same as in panel A, except that LMP1 (B) and EBNA-2 (C) mRNA were quantified relative to actin for each cell line.
    Figure Legend Snippet: BIC RNA levels are highest in EBV-positive LCL cells. (A) BIC RNA levels were assayed by RT-PCR and normalized to actin RNA levels for various cell lines (DG75 is an EBV-negative BL cell line, Raji is EBV-positive BL cell line, LCL is an EBV-positive lymphoblastoid cell line, 293T is an EBV-negative HEK cell line, ZKO is a Zta knockout EBV bacmid-positive 293 cell line, and D98 is an EBV P3HR1 strain-positive HeLa cell line). (B and C) Same as in panel A, except that LMP1 (B) and EBNA-2 (C) mRNA were quantified relative to actin for each cell line.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Knock-Out

    miR-155 and BIC are induced by EBV immortalization of primary B lymphocytes. (A) Total RNA was isolated from human peripheral blood cells from seven different donors at 10 days postinfection with EBV (EBV), at 10 days after mock infection (Mock), or immediately prior to EBV infection (Pre). RNA was analyzed for miRNA expression by using differential hybridization. miRNA expression was grouped by hierarchical cluster analysis with RNA samples in the vertical axis and by relevant miRNAs in the horizontal axis. (B) RNA was isolated from preinfected peripheral blood cell samples or from peripheral blood cell samples (1 to 7) at 10 days postinfection or at 10 days after mock infection and assayed by RT-PCR for the miR-155 precursor BIC RNA relative to cellular actin RNA. (C) Northern blot analysis of total RNA isolated from samples 4, 5, and 6 from preinfection (D0), mock-infected (−), or EBV (+)-infected peripheral blood samples (10 days postinfection) and analyzed with a probe specific for miR-155 (top panel) or control U6 snRNA (lower panel). (D) BIC RNA relative to actin RNA was measured by RT-PCR at various days (as indicated) after EBV infection or after mock infection of peripheral blood cells. (E) EBV-encoded LMP1 and EBNA2, along with BIC RNA, were measured at 10 days posttransfection relative to actin RNA using RT-PCR for EBV-infected samples from donors 4, 5, and 6.
    Figure Legend Snippet: miR-155 and BIC are induced by EBV immortalization of primary B lymphocytes. (A) Total RNA was isolated from human peripheral blood cells from seven different donors at 10 days postinfection with EBV (EBV), at 10 days after mock infection (Mock), or immediately prior to EBV infection (Pre). RNA was analyzed for miRNA expression by using differential hybridization. miRNA expression was grouped by hierarchical cluster analysis with RNA samples in the vertical axis and by relevant miRNAs in the horizontal axis. (B) RNA was isolated from preinfected peripheral blood cell samples or from peripheral blood cell samples (1 to 7) at 10 days postinfection or at 10 days after mock infection and assayed by RT-PCR for the miR-155 precursor BIC RNA relative to cellular actin RNA. (C) Northern blot analysis of total RNA isolated from samples 4, 5, and 6 from preinfection (D0), mock-infected (−), or EBV (+)-infected peripheral blood samples (10 days postinfection) and analyzed with a probe specific for miR-155 (top panel) or control U6 snRNA (lower panel). (D) BIC RNA relative to actin RNA was measured by RT-PCR at various days (as indicated) after EBV infection or after mock infection of peripheral blood cells. (E) EBV-encoded LMP1 and EBNA2, along with BIC RNA, were measured at 10 days posttransfection relative to actin RNA using RT-PCR for EBV-infected samples from donors 4, 5, and 6.

    Techniques Used: Isolation, Infection, Expressing, Hybridization, Reverse Transcription Polymerase Chain Reaction, Northern Blot

    13) Product Images from "New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes"

    Article Title: New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes

    Journal: PeerJ

    doi: 10.7717/peerj.8406

    Comparison of applied DNA isolation/amplification procedures. Numbers indicate corresponding procedures described in the text. For procedures (1), (2) and (3) all steps of DNA isolation are visualised with respect to the manufacturer’s instructions; for all of them one standard PCR protocol was applied. For procedure (4) all applied steps are shown: intake of spores, spores disruption in a Tris-EDTA buffer by vortexing (spore preparation) and SSU rDNA amplification using direct PCR.
    Figure Legend Snippet: Comparison of applied DNA isolation/amplification procedures. Numbers indicate corresponding procedures described in the text. For procedures (1), (2) and (3) all steps of DNA isolation are visualised with respect to the manufacturer’s instructions; for all of them one standard PCR protocol was applied. For procedure (4) all applied steps are shown: intake of spores, spores disruption in a Tris-EDTA buffer by vortexing (spore preparation) and SSU rDNA amplification using direct PCR.

    Techniques Used: DNA Extraction, Amplification, Polymerase Chain Reaction

    14) Product Images from "p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon"

    Article Title: p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    Journal: The Plant Journal

    doi: 10.1111/tpj.12420

    BdPMT expression levels, locations of mutations, and sequence homologies. (a) BdPMT transcript levels in WT Brachypodium tissues, as determined by qRT-PCR. Columns represent means ( n = 3), bars, standard deviations. Means were normalized to the first internode mean transcript level value, which was set to one. (b) Diagram of the BdPMT gene Bradi2g36910 (drawn to scale; 300 bp scale bar). Solid rectangles represent the two exons, connected by a 4009 bp intron. Labeled are relative locations of translational start/stop codons, Bdpmt-1 transition mutation, and the RNAi constructs. (c) Alignment of BdPMT polypeptide sequences containing the HXXXD motif with sequences from BAHD acyltransferases representing each of the five clades as delineated by phylogenetic analysis (D' Auria, 2006 ). Consensus amino acids are boxed in black. The HXXXD motif is delineated along with the position of the G that was mutated in the Bdpmt-1 mutant allele. References for the acyltransferases (delineated by accession numbers) are in D'Auria (2006) .
    Figure Legend Snippet: BdPMT expression levels, locations of mutations, and sequence homologies. (a) BdPMT transcript levels in WT Brachypodium tissues, as determined by qRT-PCR. Columns represent means ( n = 3), bars, standard deviations. Means were normalized to the first internode mean transcript level value, which was set to one. (b) Diagram of the BdPMT gene Bradi2g36910 (drawn to scale; 300 bp scale bar). Solid rectangles represent the two exons, connected by a 4009 bp intron. Labeled are relative locations of translational start/stop codons, Bdpmt-1 transition mutation, and the RNAi constructs. (c) Alignment of BdPMT polypeptide sequences containing the HXXXD motif with sequences from BAHD acyltransferases representing each of the five clades as delineated by phylogenetic analysis (D' Auria, 2006 ). Consensus amino acids are boxed in black. The HXXXD motif is delineated along with the position of the G that was mutated in the Bdpmt-1 mutant allele. References for the acyltransferases (delineated by accession numbers) are in D'Auria (2006) .

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR, Labeling, Mutagenesis, Construct

    15) Product Images from "HDAC inhibitors stimulate viral transcription by multiple mechanisms"

    Article Title: HDAC inhibitors stimulate viral transcription by multiple mechanisms

    Journal: Virology Journal

    doi: 10.1186/1743-422X-5-43

    Association of p300 with transcribing SV40 minichromosomes after HDACi treatment . Unfixed SV40 minichromosomes treated with 250 μM NaBu or 120 nM TSA were isolated from cells infected with 776 wild-type virus for 48 hours, immunoprecipitated with RNAPII, and then subjected to an ISFIP/ReChIP analysis with antibody against p300. The samples were amplified by simplex PCR with primer sets to the late region. The position of the amplification product from the wild-type 776 DNA is indicated. Lane 1, ISFIP input fraction; lane 2, ChIP with 7.5 μl of hyperacetylated histone H4 antibody (ISFIP); lane 3, ChIP with 10 μl of p300 antibody (ISFIP); lane 4, ReChIP input fraction; lane 5, ChIP with 7.5 μl of hyperacetylated histone H4 antibody.
    Figure Legend Snippet: Association of p300 with transcribing SV40 minichromosomes after HDACi treatment . Unfixed SV40 minichromosomes treated with 250 μM NaBu or 120 nM TSA were isolated from cells infected with 776 wild-type virus for 48 hours, immunoprecipitated with RNAPII, and then subjected to an ISFIP/ReChIP analysis with antibody against p300. The samples were amplified by simplex PCR with primer sets to the late region. The position of the amplification product from the wild-type 776 DNA is indicated. Lane 1, ISFIP input fraction; lane 2, ChIP with 7.5 μl of hyperacetylated histone H4 antibody (ISFIP); lane 3, ChIP with 10 μl of p300 antibody (ISFIP); lane 4, ReChIP input fraction; lane 5, ChIP with 7.5 μl of hyperacetylated histone H4 antibody.

    Techniques Used: Isolation, Infection, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    16) Product Images from "Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction"

    Article Title: Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001064

    Olfactory expression of IRs in Aplysia molluscs. (A) Top: Schematic representation of Aplysia , illustrating the location of selected sensory, neuronal and reproductive tissues used for RNA isolation and RT-PCR (adapted from [21] ). The central nervous system samples comprised pooled cerebral, pleural, buccal, pedal and abdominal ganglia. The skin samples were taken from the side of the head. Bottom: RT-PCR analysis of Aplysia IR gene expression from the indicated species and tissues. Only rhinophores from A. californica (Acal) were tested due to limited availability of animals, while rhinophore and other tissues were examined for the closely related species A. dactylomela ( Adac ) [92] . Nucleotide sequence identity of IR orthologues between these species is > 85%. Control RT-PCR corresponds to β-actin. (B) Schematic of Aplysia rhinophore showing the approximate location of the field of views of the rhinophore groove olfactory tissue in (C–E). (C,D) RNA in situ hybridisation on A. dactylomela rhinophore sections using a digoxigenin-labelled antisense RNA probe for AdacIR25a . Micrographs reveal IR25a expression (blue) in small clusters of cells of a characteristic neuronal morphology close to the sensory epithelial surface. Higher magnifications of specific cellular staining (arrowhead) are shown in the insets. The scale bars represent 100 µm. (E) Control RNA in situ hybridisation on an A. dactylomela rhinophore section with a digoxigenin-labelled sense riboprobe for AdacIR25a . No signal is apparent. The scale bar represents 100 µm.
    Figure Legend Snippet: Olfactory expression of IRs in Aplysia molluscs. (A) Top: Schematic representation of Aplysia , illustrating the location of selected sensory, neuronal and reproductive tissues used for RNA isolation and RT-PCR (adapted from [21] ). The central nervous system samples comprised pooled cerebral, pleural, buccal, pedal and abdominal ganglia. The skin samples were taken from the side of the head. Bottom: RT-PCR analysis of Aplysia IR gene expression from the indicated species and tissues. Only rhinophores from A. californica (Acal) were tested due to limited availability of animals, while rhinophore and other tissues were examined for the closely related species A. dactylomela ( Adac ) [92] . Nucleotide sequence identity of IR orthologues between these species is > 85%. Control RT-PCR corresponds to β-actin. (B) Schematic of Aplysia rhinophore showing the approximate location of the field of views of the rhinophore groove olfactory tissue in (C–E). (C,D) RNA in situ hybridisation on A. dactylomela rhinophore sections using a digoxigenin-labelled antisense RNA probe for AdacIR25a . Micrographs reveal IR25a expression (blue) in small clusters of cells of a characteristic neuronal morphology close to the sensory epithelial surface. Higher magnifications of specific cellular staining (arrowhead) are shown in the insets. The scale bars represent 100 µm. (E) Control RNA in situ hybridisation on an A. dactylomela rhinophore section with a digoxigenin-labelled sense riboprobe for AdacIR25a . No signal is apparent. The scale bar represents 100 µm.

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Sequencing, In Situ, Hybridization, Staining

    17) Product Images from "Regulating vitamin B12 biosynthesis via the cbiMCbl riboswitch in Propionibacterium strain UF1"

    Article Title: Regulating vitamin B12 biosynthesis via the cbiMCbl riboswitch in Propionibacterium strain UF1

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

    doi: 10.1073/pnas.1916576116

    cbiM Cbl riboswitch controls VB12 production. ( A ) qRT-PCR analysis of expression of cbiMNQO and cobA genes in P. UF1 incubated with various concentrations of VB12. ( B ) HPLC analysis of VB12 production by WT and SL1-deleted riboswitches within OW-operon-Δ cobA and OΔSL1-operon-Δ cobA strains. The bacteria-absorbed VB12 was excluded for this analysis. Data are representative of 3 independent experiments. Error bars indicate SEM. * P
    Figure Legend Snippet: cbiM Cbl riboswitch controls VB12 production. ( A ) qRT-PCR analysis of expression of cbiMNQO and cobA genes in P. UF1 incubated with various concentrations of VB12. ( B ) HPLC analysis of VB12 production by WT and SL1-deleted riboswitches within OW-operon-Δ cobA and OΔSL1-operon-Δ cobA strains. The bacteria-absorbed VB12 was excluded for this analysis. Data are representative of 3 independent experiments. Error bars indicate SEM. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Incubation, High Performance Liquid Chromatography

    cobA is essential for VB12 biosynthesis within P.UF1 ( A ) Proposed biosynthetic pathway for VB12 produced by P. UF1 in which cobA is responsible for converting uroporphyrinogen III to precorrin-2. ( B ) Genetic organization of P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains. cmR , chloramphenicol-resistant gene. hygB , hygromycin B. ( C ) PCR identification of P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains using primers P1/P2 and P3/P4 as shown in B . ( D ) Western blot (WB) analysis of cobA expression in P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains using mouse serum antibodies against CobA. The large surface layer protein (LspA) served as a reference control. ( E ) HPLC chromatograms of VB12 extracted from P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains. The bar graph shows the intracellular levels of VB12 in the indicated strains. Data are representative of 3 independent experiments. Error bars indicate SEM. ** P
    Figure Legend Snippet: cobA is essential for VB12 biosynthesis within P.UF1 ( A ) Proposed biosynthetic pathway for VB12 produced by P. UF1 in which cobA is responsible for converting uroporphyrinogen III to precorrin-2. ( B ) Genetic organization of P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains. cmR , chloramphenicol-resistant gene. hygB , hygromycin B. ( C ) PCR identification of P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains using primers P1/P2 and P3/P4 as shown in B . ( D ) Western blot (WB) analysis of cobA expression in P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains using mouse serum antibodies against CobA. The large surface layer protein (LspA) served as a reference control. ( E ) HPLC chromatograms of VB12 extracted from P. UF1, Δ cobA P. UF1, and C-Δ cobA P. UF1 strains. The bar graph shows the intracellular levels of VB12 in the indicated strains. Data are representative of 3 independent experiments. Error bars indicate SEM. ** P

    Techniques Used: Produced, Polymerase Chain Reaction, Western Blot, Expressing, High Performance Liquid Chromatography

    18) Product Images from "Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro"

    Article Title: Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-9-230

    Expression of TLR4 mRNA in neuronal and glial cell cultures. Neuronal, mixed glial, microglial and astrocytic primary cultures were analyzed by RT-PCR for expression of TLR4 and GAPDH mRNAs. cDNA amplification was visualized by agarose gel electrophoresis. Images shown are representative of three experiments carried out on separate cultures. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TLR4, Toll-like receptor 4.
    Figure Legend Snippet: Expression of TLR4 mRNA in neuronal and glial cell cultures. Neuronal, mixed glial, microglial and astrocytic primary cultures were analyzed by RT-PCR for expression of TLR4 and GAPDH mRNAs. cDNA amplification was visualized by agarose gel electrophoresis. Images shown are representative of three experiments carried out on separate cultures. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TLR4, Toll-like receptor 4.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    19) Product Images from "The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals"

    Article Title: The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0060135

    Biallelic Expression of DLK1 and DIO3 in Wallaby and Platypus (A) DLK1 is biallelically expressed in tammar wallaby. The imprinting status of wallaby DLK1 was determined by analyzing cDNAs shown here from three individuals (638, 788, and 2386) heterozygous for a G/A single nucleotide polymorphism (SNP) in exon 4 at 374 bp from translational start. Biallelic expression was observed in yolk sac placenta (YSM), fetal head, fetal tail, and pouch young (PY) body. Results were confirmed with three further SNPs in the 5′ UTR ( Figure S2 ). (B) DLK1 is biallelically expressed in platypus. An A/C SNP was identified in the 3′ UTR of the platypus DLK1 gene 1,323 bp from the translational start. Sequence analysis of cDNA generated from an informative platypus primary fibroblast cell line demonstrated biallelic expression. The C allele of the SNP introduces an Nla III into the region. RFLP analysis confirms biallelic expression of platypus DLK1 . (C) DIO3 is biallelically expressed in the platypus. Two polymorphisms in platypus DIO3 were identified in two different primary fibroblast cell lines—a G/C SNP and a 64 bp indel. RT-PCR analysis demonstrates biallelic expression. (D) Two polymorphisms were identified in wallaby DIO3, a CTT indel and a G/A SNP. Preferential expression was observed from the –CTT/G allele, which was particularly evident in yolk sac placenta samples. (E) Quantitative RT-PCR was used to assess the expression from each DIO3 allele in 12 different heterozygous individuals compared with a standard curve of two gDNA mixed at different ratios. Genomic DNA from all individuals was also tested and compared to the standard curve. Where more than one cDNA was analysed the data were combined and ± standard error are shown. All tissues tested displayed biased expression of the –CTT allele regardless of its parent of origin. BYS, bilaminar yolk sac; TYS, trilaminar yolk sac; YS, yolk sac; and mat, maternal gDNA. The maternal genotype for each individual is are shown in parentheses.
    Figure Legend Snippet: Biallelic Expression of DLK1 and DIO3 in Wallaby and Platypus (A) DLK1 is biallelically expressed in tammar wallaby. The imprinting status of wallaby DLK1 was determined by analyzing cDNAs shown here from three individuals (638, 788, and 2386) heterozygous for a G/A single nucleotide polymorphism (SNP) in exon 4 at 374 bp from translational start. Biallelic expression was observed in yolk sac placenta (YSM), fetal head, fetal tail, and pouch young (PY) body. Results were confirmed with three further SNPs in the 5′ UTR ( Figure S2 ). (B) DLK1 is biallelically expressed in platypus. An A/C SNP was identified in the 3′ UTR of the platypus DLK1 gene 1,323 bp from the translational start. Sequence analysis of cDNA generated from an informative platypus primary fibroblast cell line demonstrated biallelic expression. The C allele of the SNP introduces an Nla III into the region. RFLP analysis confirms biallelic expression of platypus DLK1 . (C) DIO3 is biallelically expressed in the platypus. Two polymorphisms in platypus DIO3 were identified in two different primary fibroblast cell lines—a G/C SNP and a 64 bp indel. RT-PCR analysis demonstrates biallelic expression. (D) Two polymorphisms were identified in wallaby DIO3, a CTT indel and a G/A SNP. Preferential expression was observed from the –CTT/G allele, which was particularly evident in yolk sac placenta samples. (E) Quantitative RT-PCR was used to assess the expression from each DIO3 allele in 12 different heterozygous individuals compared with a standard curve of two gDNA mixed at different ratios. Genomic DNA from all individuals was also tested and compared to the standard curve. Where more than one cDNA was analysed the data were combined and ± standard error are shown. All tissues tested displayed biased expression of the –CTT allele regardless of its parent of origin. BYS, bilaminar yolk sac; TYS, trilaminar yolk sac; YS, yolk sac; and mat, maternal gDNA. The maternal genotype for each individual is are shown in parentheses.

    Techniques Used: Expressing, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    20) Product Images from "The liver-enriched transcription factor CREB-H is a growth suppressor protein underexpressed in hepatocellular carcinoma"

    Article Title: The liver-enriched transcription factor CREB-H is a growth suppressor protein underexpressed in hepatocellular carcinoma

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki332

    CREB-H is underexpressed in human HCC tissues. Surgically resected HCC (T) and adjacent non-cancerous liver tissues (NT) from 26 patients were subjected to RNA extraction and subsequent RT–PCR using primers that amplify 286 bp cDNA fragment of human CREB-H, as well as a fragment of β-actin. Representative RT–PCR results were shown in ( A ). The normalized values of the relative quantities of the CREB-H PCR products as compared with the respective amount of β-actin were shown in a scatterplot ( B ). The lines indicate the median.
    Figure Legend Snippet: CREB-H is underexpressed in human HCC tissues. Surgically resected HCC (T) and adjacent non-cancerous liver tissues (NT) from 26 patients were subjected to RNA extraction and subsequent RT–PCR using primers that amplify 286 bp cDNA fragment of human CREB-H, as well as a fragment of β-actin. Representative RT–PCR results were shown in ( A ). The normalized values of the relative quantities of the CREB-H PCR products as compared with the respective amount of β-actin were shown in a scatterplot ( B ). The lines indicate the median.

    Techniques Used: RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    21) Product Images from "Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example"

    Article Title: Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-405

    Generation of double stranded DNA templates using paired synthetic long oligos . After 5 cycles of PCR, the products were run on 2% agarose gel. The formation of ~170 bp of product can be clearly visualized.
    Figure Legend Snippet: Generation of double stranded DNA templates using paired synthetic long oligos . After 5 cycles of PCR, the products were run on 2% agarose gel. The formation of ~170 bp of product can be clearly visualized.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    22) Product Images from "REH2 RNA Helicase in Kinetoplastid Mitochondria"

    Article Title: REH2 RNA Helicase in Kinetoplastid Mitochondria

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.051862

    REH2 expression in procyclic T. brucei . A , scheme of the C-terminal TAP tag and anti-(CBP) Western blot of total cell lysates with or without 3 days of induction with increasing tetracycline ( Tc ) concentrations. B , quantitative reverse transcription-PCR
    Figure Legend Snippet: REH2 expression in procyclic T. brucei . A , scheme of the C-terminal TAP tag and anti-(CBP) Western blot of total cell lysates with or without 3 days of induction with increasing tetracycline ( Tc ) concentrations. B , quantitative reverse transcription-PCR

    Techniques Used: Expressing, Western Blot, Polymerase Chain Reaction

    23) Product Images from "p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis"

    Article Title: p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis

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

    doi: 10.1073/pnas.1019400108

    AlaXp and a variant are expressed in mouse cells. ( A ) Schematic representation of primers used for RT-PCR analysis. For the fusion protein, four different forward primers (F1–F4) in the p23 H region were designed to ensure the detection of the
    Figure Legend Snippet: AlaXp and a variant are expressed in mouse cells. ( A ) Schematic representation of primers used for RT-PCR analysis. For the fusion protein, four different forward primers (F1–F4) in the p23 H region were designed to ensure the detection of the

    Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction

    24) Product Images from "Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases"

    Article Title: Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases

    Journal: Scientific Reports

    doi: 10.1038/srep21264

    G2/M synchronization increases targeting efficiency of NKX6.1 with ZFNs and INS with TALENs and CRISPR/Cas9. ( a) Human NKX6.1 gene structure and targeting scheme. ZFNs cleave using FokI nuclease at the 3′ end of the first exon to create DSB for insertion of a fluorescent reporter, eGFP. (b) The efficiency of targeting in H1 cells determined by counting the percentage of correctly targeted colonies compared to resistant colonies after PCR confirmation; n = 2 independent experiments. * P
    Figure Legend Snippet: G2/M synchronization increases targeting efficiency of NKX6.1 with ZFNs and INS with TALENs and CRISPR/Cas9. ( a) Human NKX6.1 gene structure and targeting scheme. ZFNs cleave using FokI nuclease at the 3′ end of the first exon to create DSB for insertion of a fluorescent reporter, eGFP. (b) The efficiency of targeting in H1 cells determined by counting the percentage of correctly targeted colonies compared to resistant colonies after PCR confirmation; n = 2 independent experiments. * P

    Techniques Used: TALENs, CRISPR, Polymerase Chain Reaction

    25) Product Images from "AS160 Phosphotyrosine-binding Domain Constructs Inhibit Insulin-stimulated GLUT4 Vesicle Fusion with the Plasma Membrane *"

    Article Title: AS160 Phosphotyrosine-binding Domain Constructs Inhibit Insulin-stimulated GLUT4 Vesicle Fusion with the Plasma Membrane *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.226092

    Analysis of the abundance of 14-3-3 isoforms in rat adipocytes and their interactions with AS160. A , quantitative real-time PCR analysis of the relative abundance of mRNA for the different known 14-3-3 isoforms in rat adipocytes. B , quantification of the 14-3-3β, -γ, and -ϵ proteins in rat adipocytes as detected by immunoblotting and quantified by comparison with a recombinant protein standard curve as illustrated in Fig. 4 . C , comparison of the relative affinity of recombinant 14-3-3 isoforms β, γ, and ϵ for AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with total cell lysate prepared from basal ( B ) or insulin ( I )-stimulated rat adipocytes. Relative amounts of AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-AS160 antibody. Results are means ± S.E. from three independent experiments and with a representative immunoblot ( D ). E , comparison of the interaction of recombinant 14-3-3 isoforms β, γ, and ϵ with phosphorylated AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with cell lysates prepared from basal or insulin-stimulated rat adipocytes. Relative amounts of phosphorylated AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-PAS antibody. Results are means ± S.E. from three independent experiments with a representative immunoblot ( F ).
    Figure Legend Snippet: Analysis of the abundance of 14-3-3 isoforms in rat adipocytes and their interactions with AS160. A , quantitative real-time PCR analysis of the relative abundance of mRNA for the different known 14-3-3 isoforms in rat adipocytes. B , quantification of the 14-3-3β, -γ, and -ϵ proteins in rat adipocytes as detected by immunoblotting and quantified by comparison with a recombinant protein standard curve as illustrated in Fig. 4 . C , comparison of the relative affinity of recombinant 14-3-3 isoforms β, γ, and ϵ for AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with total cell lysate prepared from basal ( B ) or insulin ( I )-stimulated rat adipocytes. Relative amounts of AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-AS160 antibody. Results are means ± S.E. from three independent experiments and with a representative immunoblot ( D ). E , comparison of the interaction of recombinant 14-3-3 isoforms β, γ, and ϵ with phosphorylated AS160. Equal amounts of GST-14-3-3β, -γ, or -ϵ immobilized on glutathione beads were incubated with cell lysates prepared from basal or insulin-stimulated rat adipocytes. Relative amounts of phosphorylated AS160 pulled down by the recombinant proteins were detected by immunoblotting with anti-PAS antibody. Results are means ± S.E. from three independent experiments with a representative immunoblot ( F ).

    Techniques Used: Real-time Polymerase Chain Reaction, Recombinant, Incubation

    26) Product Images from "The X-Linked Tumor Suppressor TSPX Interacts and Promotes Degradation of the Hepatitis B Viral Protein HBx via the Proteasome Pathway"

    Article Title: The X-Linked Tumor Suppressor TSPX Interacts and Promotes Degradation of the Hepatitis B Viral Protein HBx via the Proteasome Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022979

    Endogenously expressed TSPX enhances HBx degradation in 293T cells. (A) 293T cells express endogenous TSPX. Total RNA isolated from 293T cells were analyzed by RT-PCR with primer pairs for TSPX[538–693] and GAPDH using a standard technique. Control, PCR product with p3×FLAG-TSPX[ΔPro]; +RT, with reverse transcriptase; -RT, without reverse transcriptase. (B) Repression of endogenous TSPX increased the expression of HA-HBx in 293T cells. HA-HBx expression vector (50 ng/well) and DsRed-V5 expression vector (50 ng/well) were co-transfected into 293T cells with either control siRNA (4 pmol/well) or TSPX siRNA (4 pmol/well). Forty-eight hours after transfection, cells were lysed and analyzed by Western-blot using anti-V5 and anti-HA antibodies. Treatment with TSPX siRNA resulted in significant increase in HA-HBx expression (2.8 fold). (C) Effect of TSPX siRNA on TSPX expression. FLAG-TSPX[ΔPro] expression vector (0.1 µg/well) was co-transfected into 293T cells with control siRNA (4 pmol/well) or TSPX siRNA (4 pmol/well). Forty-eight hours after transfection, cells were lysed and analyzed by western-blot using anti-FLAG antibody. TSPX siRNA significantly decreased the protein level of FLAG-TSPX[ΔPro].
    Figure Legend Snippet: Endogenously expressed TSPX enhances HBx degradation in 293T cells. (A) 293T cells express endogenous TSPX. Total RNA isolated from 293T cells were analyzed by RT-PCR with primer pairs for TSPX[538–693] and GAPDH using a standard technique. Control, PCR product with p3×FLAG-TSPX[ΔPro]; +RT, with reverse transcriptase; -RT, without reverse transcriptase. (B) Repression of endogenous TSPX increased the expression of HA-HBx in 293T cells. HA-HBx expression vector (50 ng/well) and DsRed-V5 expression vector (50 ng/well) were co-transfected into 293T cells with either control siRNA (4 pmol/well) or TSPX siRNA (4 pmol/well). Forty-eight hours after transfection, cells were lysed and analyzed by Western-blot using anti-V5 and anti-HA antibodies. Treatment with TSPX siRNA resulted in significant increase in HA-HBx expression (2.8 fold). (C) Effect of TSPX siRNA on TSPX expression. FLAG-TSPX[ΔPro] expression vector (0.1 µg/well) was co-transfected into 293T cells with control siRNA (4 pmol/well) or TSPX siRNA (4 pmol/well). Forty-eight hours after transfection, cells were lysed and analyzed by western-blot using anti-FLAG antibody. TSPX siRNA significantly decreased the protein level of FLAG-TSPX[ΔPro].

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Plasmid Preparation, Transfection, Western Blot

    27) Product Images from "Self-(in)compatibility genotypes of Moroccan apricots indicate differences and similarities in the crop history of European and North African apricot germplasm"

    Article Title: Self-(in)compatibility genotypes of Moroccan apricots indicate differences and similarities in the crop history of European and North African apricot germplasm

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-13-196

    PCR products (in negative) in 55 Moroccan apricot accessions using the second intron consensus primers of Prunus S - RNase gene. M: 1 kb?+?DNA ladder; numbers refer to samples shown in Table 1 .
    Figure Legend Snippet: PCR products (in negative) in 55 Moroccan apricot accessions using the second intron consensus primers of Prunus S - RNase gene. M: 1 kb?+?DNA ladder; numbers refer to samples shown in Table 1 .

    Techniques Used: Polymerase Chain Reaction

    PCR amplification of the SFB gene to differentiate between SFB C and SFB 8 alleles in Moroccan apricots. M: 1 kb?+?DNA ladder; numbers refer to samples shown in Table 1 . Samples 18 ( S 2 S 20 ), 32 ( S 7 S 11 ) and 44 ( S 2 S 13 ) were used as negative controls to indicate the reliability of the S C / S 8 -haplotype-specific PCR.
    Figure Legend Snippet: PCR amplification of the SFB gene to differentiate between SFB C and SFB 8 alleles in Moroccan apricots. M: 1 kb?+?DNA ladder; numbers refer to samples shown in Table 1 . Samples 18 ( S 2 S 20 ), 32 ( S 7 S 11 ) and 44 ( S 2 S 13 ) were used as negative controls to indicate the reliability of the S C / S 8 -haplotype-specific PCR.

    Techniques Used: Polymerase Chain Reaction, Amplification

    28) Product Images from "Enhancement of Reproductive Heat Tolerance in Plants"

    Article Title: Enhancement of Reproductive Heat Tolerance in Plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0122933

    Photographs of RT-PCR and Western Blot analysis showing the expression of AtHSP101 and presence of HSP101 proteins in transgenic cotton lines. RT-PCR products from pollen (A) and leaf (B) tissues of transgenic line 40 plants grown in 31°C/27°C greenhouse; Western blot analysis of HSP101 for: mature pollen of transgenic line 40 (C) and null line 52 (F, AtHSP101 minus) plants grown in a greenhouse set to 31°C/27°C day/night; leaf tissues of transgenic line 40 (D) and null line 52 (G) plants grown in a greenhouse set to 31C°/27°C day/night; leaf tissues of transgenic line 40 (E) and null line 52 (H) plants grown in a hot greenhouse set to 43°C/28°C day/night temperatures.
    Figure Legend Snippet: Photographs of RT-PCR and Western Blot analysis showing the expression of AtHSP101 and presence of HSP101 proteins in transgenic cotton lines. RT-PCR products from pollen (A) and leaf (B) tissues of transgenic line 40 plants grown in 31°C/27°C greenhouse; Western blot analysis of HSP101 for: mature pollen of transgenic line 40 (C) and null line 52 (F, AtHSP101 minus) plants grown in a greenhouse set to 31°C/27°C day/night; leaf tissues of transgenic line 40 (D) and null line 52 (G) plants grown in a greenhouse set to 31C°/27°C day/night; leaf tissues of transgenic line 40 (E) and null line 52 (H) plants grown in a hot greenhouse set to 43°C/28°C day/night temperatures.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transgenic Assay

    Genomic PCR analysis of cellulose synthase and AtHSP101 in segregating transgenic cotton lines and a commercial check (Phytogen 72).
    Figure Legend Snippet: Genomic PCR analysis of cellulose synthase and AtHSP101 in segregating transgenic cotton lines and a commercial check (Phytogen 72).

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay

    29) Product Images from "A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding"

    Article Title: A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

    Journal: Journal of Virology

    doi: 10.1128/JVI.02399-16

    M21T significantly enhanced viral assembly efficiency. Full-length JFH1 RNAs containing WT or M21T NS3 were electroporated into Huh7.5.1 cells, and the cells were harvested at day 2 and day 3 postelectroporation. (A) Intracellular HCV RNA levels were analyzed by quantitative reverse transcription-PCR (RT-qPCR). Extracellular (B) and intracellular (C) infectivities were determined by titration assay. Error bars indicate standard deviations calculated from 3 independent experiments.
    Figure Legend Snippet: M21T significantly enhanced viral assembly efficiency. Full-length JFH1 RNAs containing WT or M21T NS3 were electroporated into Huh7.5.1 cells, and the cells were harvested at day 2 and day 3 postelectroporation. (A) Intracellular HCV RNA levels were analyzed by quantitative reverse transcription-PCR (RT-qPCR). Extracellular (B) and intracellular (C) infectivities were determined by titration assay. Error bars indicate standard deviations calculated from 3 independent experiments.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Titration

    30) Product Images from "Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation"

    Article Title: Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation

    Journal: eLife

    doi: 10.7554/eLife.14707

    Effect of mutation of non-aromatic residues in RBM5 OCRE on FAS alternative splicing regulation. ( A ) Mutations at position Ser468 of RBM5 OCRE domain impair the activity of the protein in FAS alternative splicing regulation ex vivo. HeLa cells were co-transfected with a FAS alternative splicing reporter and T7-RBM5 expression plasmids. RNA and proteins were isolated 24 hr after transfection. The pattern of alternative splicing was studied by RT-PCR using specific primers (PT1/PT2). Inclusion and skipping products are annotated. The asterisk indicates non-specific amplification. ( B ) Protein expression corresponding to the samples in ( A ), detected by western blot with an anti-T7 epitope antibody. ( C ) Quantification of the activity of RBM5 OCRE domain mutants on FAS alternative splicing regulation for 12 to 19 replicates, as indicated at the bottom of the histogram bars. Average and standard deviation of the percentage of inclusion is represented. T-test (two-tailed distribution, homoscedastic) results are indicated (***
    Figure Legend Snippet: Effect of mutation of non-aromatic residues in RBM5 OCRE on FAS alternative splicing regulation. ( A ) Mutations at position Ser468 of RBM5 OCRE domain impair the activity of the protein in FAS alternative splicing regulation ex vivo. HeLa cells were co-transfected with a FAS alternative splicing reporter and T7-RBM5 expression plasmids. RNA and proteins were isolated 24 hr after transfection. The pattern of alternative splicing was studied by RT-PCR using specific primers (PT1/PT2). Inclusion and skipping products are annotated. The asterisk indicates non-specific amplification. ( B ) Protein expression corresponding to the samples in ( A ), detected by western blot with an anti-T7 epitope antibody. ( C ) Quantification of the activity of RBM5 OCRE domain mutants on FAS alternative splicing regulation for 12 to 19 replicates, as indicated at the bottom of the histogram bars. Average and standard deviation of the percentage of inclusion is represented. T-test (two-tailed distribution, homoscedastic) results are indicated (***

    Techniques Used: Mutagenesis, Activity Assay, Ex Vivo, Transfection, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Standard Deviation, Two Tailed Test

    31) Product Images from "At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material"

    Article Title: At the crossroads of botanical collections and molecular genetics laboratory: a preliminary study of obtaining amplifiable DNA from moss herbarium material

    Journal: PeerJ

    doi: 10.7717/peerj.9109

    CTAB extraction test. Effect of extraction method on PCR success (%) measured as the number of positive amplicons divided by the total number of samples, before and after using Genomic DNA Clean Concentrator-10 kit. (A) ITS5 bryo- ITSC bryo, (B) ITS5D bryo- ITS4 bryo, (C) trnT-trnF , (D) rps4 , (E) ITS5 bryo- ITSC bryo, ITS5D bryo- ITS4 bryo, trnT-trnF , rps4 .
    Figure Legend Snippet: CTAB extraction test. Effect of extraction method on PCR success (%) measured as the number of positive amplicons divided by the total number of samples, before and after using Genomic DNA Clean Concentrator-10 kit. (A) ITS5 bryo- ITSC bryo, (B) ITS5D bryo- ITS4 bryo, (C) trnT-trnF , (D) rps4 , (E) ITS5 bryo- ITSC bryo, ITS5D bryo- ITS4 bryo, trnT-trnF , rps4 .

    Techniques Used: Polymerase Chain Reaction

    32) Product Images from "Enterovirus 71 induces neural cell apoptosis and autophagy through promoting ACOX1 downregulation and ROS generation"

    Article Title: Enterovirus 71 induces neural cell apoptosis and autophagy through promoting ACOX1 downregulation and ROS generation

    Journal: Virulence

    doi: 10.1080/21505594.2020.1766790

    EV71 attenuates ACOX1 protein expression through 3D. (a–d) RD cells were infected with EV71 at an MOI of 2 for different times (a) and (c) or at different MOIs for 12 h (b) and (d). The ACOX1 mRNA (A and B, top panels) and EV71 VP1 mRNA ((a) and (b), bottom panels) were measured by qRT-PCR and normalized to GAPDH mRNA. The ACOX1 and EV71 VP1 proteins in WCLs were determined by Western blot, GAPDH was used as loading control (c) and (d). (e) SK-N-SH cells were infected with EV71 at different MOIs for 48 h. (f) U251 cells were infected with EV71 at different MOIs for 24 h. (g) SK-N-SH cells were infected with EV71 at an MOI of 2 for different times, the viability of cells was measured by CCK8 assay. (h) RD cells were transfected with pFlag-ACOX1 for 24 h, and then infected with EV71 at different MOIs for 12 h. (i) RD cells were transfected with vector or pHA-3D at different concentrations for 36 h. (j) Control U251 cells and stable U251 cells expressing EV71 3D were treated with DMSO, CQ (10 μM), or MG132 (20 μM) for 10 h. (k) HEK293 T cells were co-transfected with pFlag-ACOX1 along with pHA-3D. Cells were treated with or without MG132 (20 μM) for 10 hours before harvest. (l) Control U251 cells and stable U251 cells expressing EV71 3D were analyzed by Western blot.
    Figure Legend Snippet: EV71 attenuates ACOX1 protein expression through 3D. (a–d) RD cells were infected with EV71 at an MOI of 2 for different times (a) and (c) or at different MOIs for 12 h (b) and (d). The ACOX1 mRNA (A and B, top panels) and EV71 VP1 mRNA ((a) and (b), bottom panels) were measured by qRT-PCR and normalized to GAPDH mRNA. The ACOX1 and EV71 VP1 proteins in WCLs were determined by Western blot, GAPDH was used as loading control (c) and (d). (e) SK-N-SH cells were infected with EV71 at different MOIs for 48 h. (f) U251 cells were infected with EV71 at different MOIs for 24 h. (g) SK-N-SH cells were infected with EV71 at an MOI of 2 for different times, the viability of cells was measured by CCK8 assay. (h) RD cells were transfected with pFlag-ACOX1 for 24 h, and then infected with EV71 at different MOIs for 12 h. (i) RD cells were transfected with vector or pHA-3D at different concentrations for 36 h. (j) Control U251 cells and stable U251 cells expressing EV71 3D were treated with DMSO, CQ (10 μM), or MG132 (20 μM) for 10 h. (k) HEK293 T cells were co-transfected with pFlag-ACOX1 along with pHA-3D. Cells were treated with or without MG132 (20 μM) for 10 hours before harvest. (l) Control U251 cells and stable U251 cells expressing EV71 3D were analyzed by Western blot.

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transfection, Plasmid Preparation

    EV71 infection and ACOX1 knockdown promote neural cell death through inducing ROS and attenuating the DJ-1/NRF2/HO-1 pathway. (a) RD cells were infected with EV71 at the indicated MOIs for 18 h. SK-N-SH cells were infected with EV71 at the indicated MOIs for 48 h. U251 cells were infected with EV71 at the indicated MOIs for 24 h. Cellular ROS level was tested using a ROS detection kit. (b) Cellular ROS levels in stable ACOX1 knockdown RD cells, stable ACOX1 knockdown SK-N-SH cells, and stable ACOX1 knockdown U251 cells were tested. (c) RD cells were infected with EV71 at different MOIs for 12 h. U251 cells were infected with EV71 at an MOI of 2 for different times. (d) Stable ACOX1 knockdown cells were subjected to Western blot analysis. (e) RD cells were infected with EV71 at an MOI of 2 for different times. SK-N-SH cells were infected with EV71 at an MOI of 2 for different times. U251 cells were infected with EV71 at an MOI of 2 for different times. (f) Stable EV71 3D expressing cells were subjected to Western blot analysis. (g) Stable ACOX1 knockdown cells were subjected to Western blot analysis. (h) RD cells, and U251 cells were transfected with vector or plasmid expressing EV71 3D protein, 48 hours after transfection, the mRNA levels of NRF2, and DJ-1 were measured by qRT-PCR and normalized to GAPDH mRNA.
    Figure Legend Snippet: EV71 infection and ACOX1 knockdown promote neural cell death through inducing ROS and attenuating the DJ-1/NRF2/HO-1 pathway. (a) RD cells were infected with EV71 at the indicated MOIs for 18 h. SK-N-SH cells were infected with EV71 at the indicated MOIs for 48 h. U251 cells were infected with EV71 at the indicated MOIs for 24 h. Cellular ROS level was tested using a ROS detection kit. (b) Cellular ROS levels in stable ACOX1 knockdown RD cells, stable ACOX1 knockdown SK-N-SH cells, and stable ACOX1 knockdown U251 cells were tested. (c) RD cells were infected with EV71 at different MOIs for 12 h. U251 cells were infected with EV71 at an MOI of 2 for different times. (d) Stable ACOX1 knockdown cells were subjected to Western blot analysis. (e) RD cells were infected with EV71 at an MOI of 2 for different times. SK-N-SH cells were infected with EV71 at an MOI of 2 for different times. U251 cells were infected with EV71 at an MOI of 2 for different times. (f) Stable EV71 3D expressing cells were subjected to Western blot analysis. (g) Stable ACOX1 knockdown cells were subjected to Western blot analysis. (h) RD cells, and U251 cells were transfected with vector or plasmid expressing EV71 3D protein, 48 hours after transfection, the mRNA levels of NRF2, and DJ-1 were measured by qRT-PCR and normalized to GAPDH mRNA.

    Techniques Used: Infection, Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

    33) Product Images from "Transcription Factor Glis3, a Novel Critical Player in the Regulation of Pancreatic ?-Cell Development and Insulin Gene Expression ▿"

    Article Title: Transcription Factor Glis3, a Novel Critical Player in the Regulation of Pancreatic ?-Cell Development and Insulin Gene Expression ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01259-09

    Glis3 functions as a transcriptional regulator of insulin gene expression. (A) Schematic representation of potential Glis-BS and other enhancers in the murine and rat Ins2 and human INS 5′ regulatory regions. The scale at the top is in base pairs relative to the transcriptional start site (represented as +1). (B) Comparison of the nucleotide sequences of the consensus Glis-BS with the two putative Glis-BS in the mouse Ins2 and human INS proximal promoter regions. (C) Two putative Glis-BS-containing regions in the mouse insulin 2 promoter (Glis-BS1 at −84 to −109 and Glis-BS2 at −253 to −276) and their respective mutant nucleotides were labeled and used as probes in an EMSA. Competition was carried out with 5-fold (+) and 25-fold (++) excesses of unlabeled oligonucleotides as indicated. Arrow indicates the Glis3-Glis-BS complex. (D, E) Binding of Glis3 to 32 P-labeled consensus Glis-BS was examined in the absence or presence of unlabeled consensus Glis-BS, mouse Glis-BS1, and mGlis-BS2 sites, their mutants (mut), or the corresponding human INS Glis-BS. (F) INS-1 (832/13) cells were cotransfected with the pGL4.10 empty vector, mIns2-696-Luc, or hINS-700-Luc and either the p3xFlag-CMV-10 empty vector, p3xFlag-CMV-Glis3, or p3xFlag-CMV-Glis3ΔC748 as indicated. After 24 h, cells were assayed for luciferase and β-galactosidase activities and the relative Luc activity was calculated and plotted. Each bar represents the mean ± the standard error of the mean. (G) INS-1 (832/13) cells were cotransfected with the pGL4.10 empty vector, mIns2(−696)-Luc, or the specified mIns2(−696)-Luc mutant and either p3xFlag-CMV-10, p3xFlag-CMV-Glis3, or p3xFlag-CMV-Glis3ΔN302 as indicated. Relative Luc activity was calculated and plotted. In the insert, shaded boxes indicate Glis-BS and X indicates mutated Glis-BS. (H) INS-1 (832/13) cells were transiently transfected with p3xFlag-CMV-Glis3 or p3xFlag-CMV-Glis3ΔN302 and 48 h later analyzed by QRT-PCR for the expression of rat Ins1 and Ins2 and mouse Glis3.
    Figure Legend Snippet: Glis3 functions as a transcriptional regulator of insulin gene expression. (A) Schematic representation of potential Glis-BS and other enhancers in the murine and rat Ins2 and human INS 5′ regulatory regions. The scale at the top is in base pairs relative to the transcriptional start site (represented as +1). (B) Comparison of the nucleotide sequences of the consensus Glis-BS with the two putative Glis-BS in the mouse Ins2 and human INS proximal promoter regions. (C) Two putative Glis-BS-containing regions in the mouse insulin 2 promoter (Glis-BS1 at −84 to −109 and Glis-BS2 at −253 to −276) and their respective mutant nucleotides were labeled and used as probes in an EMSA. Competition was carried out with 5-fold (+) and 25-fold (++) excesses of unlabeled oligonucleotides as indicated. Arrow indicates the Glis3-Glis-BS complex. (D, E) Binding of Glis3 to 32 P-labeled consensus Glis-BS was examined in the absence or presence of unlabeled consensus Glis-BS, mouse Glis-BS1, and mGlis-BS2 sites, their mutants (mut), or the corresponding human INS Glis-BS. (F) INS-1 (832/13) cells were cotransfected with the pGL4.10 empty vector, mIns2-696-Luc, or hINS-700-Luc and either the p3xFlag-CMV-10 empty vector, p3xFlag-CMV-Glis3, or p3xFlag-CMV-Glis3ΔC748 as indicated. After 24 h, cells were assayed for luciferase and β-galactosidase activities and the relative Luc activity was calculated and plotted. Each bar represents the mean ± the standard error of the mean. (G) INS-1 (832/13) cells were cotransfected with the pGL4.10 empty vector, mIns2(−696)-Luc, or the specified mIns2(−696)-Luc mutant and either p3xFlag-CMV-10, p3xFlag-CMV-Glis3, or p3xFlag-CMV-Glis3ΔN302 as indicated. Relative Luc activity was calculated and plotted. In the insert, shaded boxes indicate Glis-BS and X indicates mutated Glis-BS. (H) INS-1 (832/13) cells were transiently transfected with p3xFlag-CMV-Glis3 or p3xFlag-CMV-Glis3ΔN302 and 48 h later analyzed by QRT-PCR for the expression of rat Ins1 and Ins2 and mouse Glis3.

    Techniques Used: Expressing, Mutagenesis, Labeling, Binding Assay, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR

    34) Product Images from "Efficient generation and rapid isolation via stoplight recombination of Herpes simplex viruses expressing model antigenic and immunological epitopes"

    Article Title: Efficient generation and rapid isolation via stoplight recombination of Herpes simplex viruses expressing model antigenic and immunological epitopes

    Journal: Journal of virological methods

    doi: 10.1016/j.jviromet.2011.10.009

    Diagnostic PCR of recombinant viruses after plaque purification rounds 1 or 3 to determine presence of immunological epitopes and absence of gK-null contaminating virus. (A) Primer pairs P5′/P1 were utilized to detect the rescue of the UL53/gK
    Figure Legend Snippet: Diagnostic PCR of recombinant viruses after plaque purification rounds 1 or 3 to determine presence of immunological epitopes and absence of gK-null contaminating virus. (A) Primer pairs P5′/P1 were utilized to detect the rescue of the UL53/gK

    Techniques Used: Diagnostic Assay, Polymerase Chain Reaction, Recombinant, Purification

    35) Product Images from "Control of Phage Bxb1 Excision by a Novel Recombination Directionality FactorA Novel Phage Protein Mediates the Virus's Removal from Bacterial Chromosomes"

    Article Title: Control of Phage Bxb1 Excision by a Novel Recombination Directionality FactorA Novel Phage Protein Mediates the Virus's Removal from Bacterial Chromosomes

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0040186

    Confirmation of Site-Specific Recombination and Bxb1 Integrase Dependence (A) DNA from five sucrose-resistant transformants of the excision tester strain transformed with either pPG1 (empty vector), pPGX1, or pPGX6b was examined by PCR for the presence of attB, attL, and attR . Transformation with pPGX1 and pPGX6b leads to the presence of a product amplified with attB -specific primers; no product is observed using primers that amplify attL and attR. DNA from a Bxb1 lysogen and from clones transformed with the empty vector that were used as controls show a product corresponding to attL and attR, whereas DNA from wild-type mc 2 155 shows the presence of a product corresponding to attB . (B) An int − excision tester strain was created (as in Figure 1 A) using transient expression of gpInt. The int − tester strain was then transformed with pAIK5 (gpInt alone), pX6b (gp47 alone), or pAIK5+47 (gpInt + gp47), and the frequency of suc R colonies (and therefore excision) determined. CFU, colony-forming units
    Figure Legend Snippet: Confirmation of Site-Specific Recombination and Bxb1 Integrase Dependence (A) DNA from five sucrose-resistant transformants of the excision tester strain transformed with either pPG1 (empty vector), pPGX1, or pPGX6b was examined by PCR for the presence of attB, attL, and attR . Transformation with pPGX1 and pPGX6b leads to the presence of a product amplified with attB -specific primers; no product is observed using primers that amplify attL and attR. DNA from a Bxb1 lysogen and from clones transformed with the empty vector that were used as controls show a product corresponding to attL and attR, whereas DNA from wild-type mc 2 155 shows the presence of a product corresponding to attB . (B) An int − excision tester strain was created (as in Figure 1 A) using transient expression of gpInt. The int − tester strain was then transformed with pAIK5 (gpInt alone), pX6b (gp47 alone), or pAIK5+47 (gpInt + gp47), and the frequency of suc R colonies (and therefore excision) determined. CFU, colony-forming units

    Techniques Used: Transformation Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Expressing

    36) Product Images from "The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology"

    Article Title: The Escherichia coli K-12 ORFeome: a resource for comparative molecular microbiology

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-470

    Vectors and cloning strategy . ( A ) pAZ677 Cm R . ( B ) Construction of pENTR/Zeo by BP recombination with pDONR™/Zeo. The resulting vector has attL1, attL2 sites and two SfiI sites bordering the Cm R fragment. ( C ) The E. coli ORFs of pCA24N were then transferred by SfiI digestion, gel-fractionated and ligated into SfiI-digested pENTR/Zeo vector. The positive clones are selected for Zeocin resistance and Chloramphenicol sensitivity, and are validated by PCR and DNA sequencing (see methods).
    Figure Legend Snippet: Vectors and cloning strategy . ( A ) pAZ677 Cm R . ( B ) Construction of pENTR/Zeo by BP recombination with pDONR™/Zeo. The resulting vector has attL1, attL2 sites and two SfiI sites bordering the Cm R fragment. ( C ) The E. coli ORFs of pCA24N were then transferred by SfiI digestion, gel-fractionated and ligated into SfiI-digested pENTR/Zeo vector. The positive clones are selected for Zeocin resistance and Chloramphenicol sensitivity, and are validated by PCR and DNA sequencing (see methods).

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, DNA Sequencing

    37) Product Images from "Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation"

    Article Title: Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.02.103

    Genetic Requirements of Type I-D CRISPR Adaptation (A) Overview of the type I-D CRISPR-Cas locus as found on the Synechocystis 6803 pSYSA megaplasmid. The putative adaptation module consisting of cas4-1-2 is highlighted in light brown. Downstream of cas2 C). A). (C) Co-expression of cas1 and cas2 is necessary and sufficient for the integration of new spacers. (D) Assessing spacer integration in WT E. coli K12 and different recBCD mutant backgrounds in the presence or absence of cas4 . The presence of cas4 enhances spacer integration in the ΔrecB and ΔrecC genotypes, while spacer integration is below the detection limit of this PCR (described in B) in the ΔrecD mutant regardless of the presence of cas4 .
    Figure Legend Snippet: Genetic Requirements of Type I-D CRISPR Adaptation (A) Overview of the type I-D CRISPR-Cas locus as found on the Synechocystis 6803 pSYSA megaplasmid. The putative adaptation module consisting of cas4-1-2 is highlighted in light brown. Downstream of cas2 C). A). (C) Co-expression of cas1 and cas2 is necessary and sufficient for the integration of new spacers. (D) Assessing spacer integration in WT E. coli K12 and different recBCD mutant backgrounds in the presence or absence of cas4 . The presence of cas4 enhances spacer integration in the ΔrecB and ΔrecC genotypes, while spacer integration is below the detection limit of this PCR (described in B) in the ΔrecD mutant regardless of the presence of cas4 .

    Techniques Used: CRISPR, Expressing, Mutagenesis, Polymerase Chain Reaction

    38) Product Images from "Relationship between promoter methylation tissue expression of MGMT gene in ovarian cancer"

    Article Title: Relationship between promoter methylation tissue expression of MGMT gene in ovarian cancer

    Journal: The Indian Journal of Medical Research

    doi:

    Methylation analysis of MGMT gene. Agarose gel showing representative product of MSP analysis of MGMT gene in epithelial ovarian tumours. In each case, CpGenome universal methylated genomic DNA was used as a positive (+ve) control for methylated alleles and peripheral blood mononuclear cell (PBMC) DNA from normal healthy subjects as positive control for unmethylated alleles. PCR products in lane UM indicate the presence of an unmethylated allele, whereas PCR products in lane M indicate the presence of a methylated allele. C004, C027 are carcinomas, B003 is benign adenoma and N001 is a normal tissue. No template control was used as a negative control.
    Figure Legend Snippet: Methylation analysis of MGMT gene. Agarose gel showing representative product of MSP analysis of MGMT gene in epithelial ovarian tumours. In each case, CpGenome universal methylated genomic DNA was used as a positive (+ve) control for methylated alleles and peripheral blood mononuclear cell (PBMC) DNA from normal healthy subjects as positive control for unmethylated alleles. PCR products in lane UM indicate the presence of an unmethylated allele, whereas PCR products in lane M indicate the presence of a methylated allele. C004, C027 are carcinomas, B003 is benign adenoma and N001 is a normal tissue. No template control was used as a negative control.

    Techniques Used: Methylation, Agarose Gel Electrophoresis, Positive Control, Polymerase Chain Reaction, Negative Control

    39) Product Images from "Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis"

    Article Title: Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0015295

    Construction of a lacZ reporter system. The full-length lacZ gene obtained by PCR was used to replace lacZα on pCM62. The P lac promoter was then removed from the resultant plasmid pTP325, resulting in the final plasmid pTP327.
    Figure Legend Snippet: Construction of a lacZ reporter system. The full-length lacZ gene obtained by PCR was used to replace lacZα on pCM62. The P lac promoter was then removed from the resultant plasmid pTP325, resulting in the final plasmid pTP327.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

    40) Product Images from "p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis"

    Article Title: p23H implicated as cis/trans regulator of AlaXp-directed editing for mammalian cell homeostasis

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

    doi: 10.1073/pnas.1019400108

    AlaXp and a variant are expressed in mouse cells. ( A ) Schematic representation of primers used for RT-PCR analysis. For the fusion protein, four different forward primers (F1–F4) in the p23 H region were designed to ensure the detection of the
    Figure Legend Snippet: AlaXp and a variant are expressed in mouse cells. ( A ) Schematic representation of primers used for RT-PCR analysis. For the fusion protein, four different forward primers (F1–F4) in the p23 H region were designed to ensure the detection of the

    Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction

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    BAC Assay:

    Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres
    Article Snippet: .. PCR amplification and labeling of chromatin DNA For BAC and PCR microarrays, the CENP-A input and immunocomplexes were amplified using ligation-mediated aminoallyl-dUTP (Sigma) PCR, as described by Alonso and coworkers [ ]. .. For CENP-C, and CENP-H input and immunocomplexes, 25 ng of sonicated DNA were repaired with 2.4 units of Kinase, 8 units of Klenow, and 8 units of T4 polymerase (NEB, Beverly, MA, USA) for 1 hour at 37°C, and ligation-mediated PCR was carried out with 3 ng, as described by Alonso and coworkers [ ].

    Ligation:

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division
    Article Snippet: .. CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ). .. All recombinant proteins were expressed using E.coli BL21 codon plus-RIL strain (Agilent Technologies).

    Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres
    Article Snippet: .. PCR amplification and labeling of chromatin DNA For BAC and PCR microarrays, the CENP-A input and immunocomplexes were amplified using ligation-mediated aminoallyl-dUTP (Sigma) PCR, as described by Alonso and coworkers [ ]. .. For CENP-C, and CENP-H input and immunocomplexes, 25 ng of sonicated DNA were repaired with 2.4 units of Kinase, 8 units of Klenow, and 8 units of T4 polymerase (NEB, Beverly, MA, USA) for 1 hour at 37°C, and ligation-mediated PCR was carried out with 3 ng, as described by Alonso and coworkers [ ].

    Labeling:

    Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres
    Article Snippet: .. PCR amplification and labeling of chromatin DNA For BAC and PCR microarrays, the CENP-A input and immunocomplexes were amplified using ligation-mediated aminoallyl-dUTP (Sigma) PCR, as described by Alonso and coworkers [ ]. .. For CENP-C, and CENP-H input and immunocomplexes, 25 ng of sonicated DNA were repaired with 2.4 units of Kinase, 8 units of Klenow, and 8 units of T4 polymerase (NEB, Beverly, MA, USA) for 1 hour at 37°C, and ligation-mediated PCR was carried out with 3 ng, as described by Alonso and coworkers [ ].

    Polymerase Chain Reaction:

    Article Title: Mechanistically Distinct Mouse Models for CRX-Associated Retinopathy
    Article Snippet: .. PCR amplification was performed using Jumpstart RedTaq (Sigma-Aldrich). .. Primer sets ( ) are as follows: For all mice: neo (Neo-F/R) and Crx (Total Crx-F/R); for E168d2 colony: WT Crx allele (E168d2 WT-F, E168d2-R), E168d2 allele (E168d2 Mut-F, E168d2-R); for R90W colony: WT Crx allele (R90W WT-R, R90W-R), R90W allele (R90W Mut-F, R90W-R).

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells
    Article Snippet: .. Primers used for RT-PCR of HCV 5′ NTR, GAPDH, and actin mRNAs, as well as for PCR amplification of full-length HCV 3′ NTR and 5′ NTR and their specific fragments were purchased from Sigma-Aldrich (St. Louis, USA). .. Primary antibodies against HCV NS5B, Staufen1 and protein kinase PKR were obtained from Santa Cruz Biotechnology.

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division
    Article Snippet: .. CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ). .. All recombinant proteins were expressed using E.coli BL21 codon plus-RIL strain (Agilent Technologies).

    Article Title: Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres
    Article Snippet: .. PCR amplification and labeling of chromatin DNA For BAC and PCR microarrays, the CENP-A input and immunocomplexes were amplified using ligation-mediated aminoallyl-dUTP (Sigma) PCR, as described by Alonso and coworkers [ ]. .. For CENP-C, and CENP-H input and immunocomplexes, 25 ng of sonicated DNA were repaired with 2.4 units of Kinase, 8 units of Klenow, and 8 units of T4 polymerase (NEB, Beverly, MA, USA) for 1 hour at 37°C, and ligation-mediated PCR was carried out with 3 ng, as described by Alonso and coworkers [ ].

    Article Title: Interleukin-8/CXCL8 is a growth factor for human lung cancer cells
    Article Snippet: .. A 0.5 μ g portion of total RNA was reverse-transcribed for subsequent PCR amplification for each pair of primers in a volume of 10 μ l, including 10 U of enhanced AMV reverse transcriptase (Sigma, Poole, Dorset, UK), 20 U of RNase inhibitor (Sigma), 0.5 μ g of oligo(dT)15 primer, 0.5 nmol of each dNTP and 1 × first-strand buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 8 mM MgCl2 , 1 mM dithiothreitol) provided by Sigma. .. The reaction was incubated at 45°C for 50 min. A 10 μ l portion of the RT products was then brought to a volume of 50 μ l containing 0.2 nmol of each dNTP, 1 U of Taq polymerase (Sigma), 0.1 μ g of both the upstream and downstream PCR primers and 1 × PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl2 , 0.01% gelatin) provided by Sigma.

    Article Title: Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis
    Article Snippet: .. One microgram of the total cellular RNA was then reverse-transcribed into cDNA for PCR amplification using a kit from Sigma. .. The primer sequences used for PCR have been listed in Table .

    Article Title: The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins
    Article Snippet: .. Plasmids for expression of Raf and Rep2 truncations as Trx-fusion proteins were created by PCR amplification of the RAF ORF and relevant portions of the REP2 ORF (codons 199–296, and 232–296) with flanking EcoRI and SalI sites and insertion in EcoRI/SalI-digested vector pET32 (Novagen), producing plasmids pET32-RAF, pET32-rep2199–296 and pET32-rep2232–296 . .. Two-hybrid assays Protein–protein association was assayed in a cir0 yeast strain (CTMD/3a) with eight copies of the LexA operator sequence in the basal promoter region upstream of a lacZ reporter gene integrated in the genome at the URA3 locus ( ).

    Article Title: Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens
    Article Snippet: .. N-terminal His-tagged wild-type and D52E ChvI were constructed by PCR amplification of the full-length wild-type or D52E ChvI with flanking Nde I/Xho I restriction sites and cloning into pET28a (Novagen). .. For the constructs used for yeast two-hybrid, various exoR ORFs were PCR-amplified (by primers AD ExoR F & AD ExoR R), digested (Nde I/Xho I), and cloned into the Nde I/Xho I sites of pGADT7 for N-terminal fusion to the activation domain (AD), pGADT7-ExoR, pGADT7-ExoRG73C , pGADT7-ExoRS153Y and pGADT7-ExoRG73C/S153Y .

    Generated:

    Article Title: A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division
    Article Snippet: .. CDKG1 antibody generation pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously ( ). .. All recombinant proteins were expressed using E.coli BL21 codon plus-RIL strain (Agilent Technologies).

    Construct:

    Article Title: Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens
    Article Snippet: .. N-terminal His-tagged wild-type and D52E ChvI were constructed by PCR amplification of the full-length wild-type or D52E ChvI with flanking Nde I/Xho I restriction sites and cloning into pET28a (Novagen). .. For the constructs used for yeast two-hybrid, various exoR ORFs were PCR-amplified (by primers AD ExoR F & AD ExoR R), digested (Nde I/Xho I), and cloned into the Nde I/Xho I sites of pGADT7 for N-terminal fusion to the activation domain (AD), pGADT7-ExoR, pGADT7-ExoRG73C , pGADT7-ExoRS153Y and pGADT7-ExoRG73C/S153Y .

    Expressing:

    Article Title: The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins
    Article Snippet: .. Plasmids for expression of Raf and Rep2 truncations as Trx-fusion proteins were created by PCR amplification of the RAF ORF and relevant portions of the REP2 ORF (codons 199–296, and 232–296) with flanking EcoRI and SalI sites and insertion in EcoRI/SalI-digested vector pET32 (Novagen), producing plasmids pET32-RAF, pET32-rep2199–296 and pET32-rep2232–296 . .. Two-hybrid assays Protein–protein association was assayed in a cir0 yeast strain (CTMD/3a) with eight copies of the LexA operator sequence in the basal promoter region upstream of a lacZ reporter gene integrated in the genome at the URA3 locus ( ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells
    Article Snippet: .. Primers used for RT-PCR of HCV 5′ NTR, GAPDH, and actin mRNAs, as well as for PCR amplification of full-length HCV 3′ NTR and 5′ NTR and their specific fragments were purchased from Sigma-Aldrich (St. Louis, USA). .. Primary antibodies against HCV NS5B, Staufen1 and protein kinase PKR were obtained from Santa Cruz Biotechnology.

    Plasmid Preparation:

    Article Title: The yeast 2-μm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins
    Article Snippet: .. Plasmids for expression of Raf and Rep2 truncations as Trx-fusion proteins were created by PCR amplification of the RAF ORF and relevant portions of the REP2 ORF (codons 199–296, and 232–296) with flanking EcoRI and SalI sites and insertion in EcoRI/SalI-digested vector pET32 (Novagen), producing plasmids pET32-RAF, pET32-rep2199–296 and pET32-rep2232–296 . .. Two-hybrid assays Protein–protein association was assayed in a cir0 yeast strain (CTMD/3a) with eight copies of the LexA operator sequence in the basal promoter region upstream of a lacZ reporter gene integrated in the genome at the URA3 locus ( ).

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    Millipore 20 kb fragments
    20 Kb Fragments, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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