Structured Review

TaKaRa egfp orf
Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. <t>EGFP</t> was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 <t>ORF.</t> The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.
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Images

1) Product Images from "UL40-mediated NK evasion during productive infection with human cytomegalovirus"

Article Title: UL40-mediated NK evasion during productive infection with human cytomegalovirus

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

doi: 10.1073/pnas.112680099

Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. EGFP was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 ORF. The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.
Figure Legend Snippet: Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. EGFP was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 ORF. The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Generated, Positive Control, Infection, Plaque Assay

2) Product Images from "Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs"

Article Title: Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs

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

doi:

Targeted disruption of the mouse G zα gene. ( A ) Knockout construct. Primers K1, K2, K3, and K4 were used to generate the targeting vector by PCR. The positive selection marker (PGK.Neo) and the negative selection marker (PGK.TK) are indicated. The ORF containing the first two-thirds of the coding region is replaced in-frame by EGFP. ( B ) PCR genotyping of mouse genomic DNA using the primer pairs indicated. ( C ) Immunoblots with rabbit polyclonal antibodies that recognize G zα , G i1α /G i2α , G qα , G sα , G oα , and four isoforms of G β . Note that G oα is normally expressed in brain, but not in platelets. The blots were stripped and reprobed with anti-ERK1/2 as a loading control. ( D ) FACS analysis of platelet-rich plasma.
Figure Legend Snippet: Targeted disruption of the mouse G zα gene. ( A ) Knockout construct. Primers K1, K2, K3, and K4 were used to generate the targeting vector by PCR. The positive selection marker (PGK.Neo) and the negative selection marker (PGK.TK) are indicated. The ORF containing the first two-thirds of the coding region is replaced in-frame by EGFP. ( B ) PCR genotyping of mouse genomic DNA using the primer pairs indicated. ( C ) Immunoblots with rabbit polyclonal antibodies that recognize G zα , G i1α /G i2α , G qα , G sα , G oα , and four isoforms of G β . Note that G oα is normally expressed in brain, but not in platelets. The blots were stripped and reprobed with anti-ERK1/2 as a loading control. ( D ) FACS analysis of platelet-rich plasma.

Techniques Used: Knock-Out, Construct, Plasmid Preparation, Polymerase Chain Reaction, Selection, Marker, Western Blot, FACS

3) Product Images from "Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition"

Article Title: Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition

Journal: Scientific Reports

doi: 10.1038/s41598-018-31256-1

Modulation of rostro-caudal and dorso-ventral axis specification induces the development of PHOX2B -expressing NCCs from hPSCs. ( a ) A diagram of the culture conditions for the modification of rostro-caudal and dorso-ventral axis specification of hPSCs. ( b ) A heat map image showing the percentage of eGFP + cells on day 10 of differentiation using KhES1 PHOX2B::eGFP under various conditions. ( c ) Representative FCM plots of day 10 KhES1 PHOX2B::eGFP-derived aggregates under the indicated conditions (i–iv) of ( b ). ( d ) RT-PCR analyses for PHOX2B , SOX1 , PAX6 , SOX10 , FOXD3 , HOXB1 , HOXB2 , HOXB4 , HOXB6 , HOXB8 and HOXC9 in day 10 aggregates under conditions (i–iv). The right diagram shows the expression pattern of HOX genes in the rhombomere (r1–8) and the spinal cord (cervical and thoracic) region. SB = SB431542, CHIR = CHIR 99021, RA = retinoic acid, Pur = Purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord.
Figure Legend Snippet: Modulation of rostro-caudal and dorso-ventral axis specification induces the development of PHOX2B -expressing NCCs from hPSCs. ( a ) A diagram of the culture conditions for the modification of rostro-caudal and dorso-ventral axis specification of hPSCs. ( b ) A heat map image showing the percentage of eGFP + cells on day 10 of differentiation using KhES1 PHOX2B::eGFP under various conditions. ( c ) Representative FCM plots of day 10 KhES1 PHOX2B::eGFP-derived aggregates under the indicated conditions (i–iv) of ( b ). ( d ) RT-PCR analyses for PHOX2B , SOX1 , PAX6 , SOX10 , FOXD3 , HOXB1 , HOXB2 , HOXB4 , HOXB6 , HOXB8 and HOXC9 in day 10 aggregates under conditions (i–iv). The right diagram shows the expression pattern of HOX genes in the rhombomere (r1–8) and the spinal cord (cervical and thoracic) region. SB = SB431542, CHIR = CHIR 99021, RA = retinoic acid, Pur = Purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord.

Techniques Used: Expressing, Modification, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells"

Article Title: EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

Journal: International Journal of Inflammation

doi: 10.1155/2015/301562

Overexpression of EOLA1 prevents LPS-induced VCAM-1 production in ECV304 cells. ECV304 cells were transfected with pOPRSVI-EOLA1-EGFP and pCMV-LacI plasmids and induced by IPTG. (a) Overexpression of EOLA1 was demonstrated by Western blotting analysis with anti-EOLA1 antibody after adding IPTG for 48 h. (b) The induced cells with IPTG were tested for their ability to produce VCAM-1 upon LPS stimulation (100 ng/mL) for 6 h using a commercial ELISA kit. Data represent the mean ± SD of three independent experiments ( ∗ p
Figure Legend Snippet: Overexpression of EOLA1 prevents LPS-induced VCAM-1 production in ECV304 cells. ECV304 cells were transfected with pOPRSVI-EOLA1-EGFP and pCMV-LacI plasmids and induced by IPTG. (a) Overexpression of EOLA1 was demonstrated by Western blotting analysis with anti-EOLA1 antibody after adding IPTG for 48 h. (b) The induced cells with IPTG were tested for their ability to produce VCAM-1 upon LPS stimulation (100 ng/mL) for 6 h using a commercial ELISA kit. Data represent the mean ± SD of three independent experiments ( ∗ p

Techniques Used: Over Expression, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

Knockdown of EOLA1 with shRNA inhibits the expression of MT2A and increases the LPS-induced VCAM-1 production in ECV304 cells. (a) ECV304 cells were transfected with control or EOLA1 shRNA for 48 h. The cells were lysed and analyzed by Western blotting with anti-EOLA1 or anti-MT2A antibodies, β -actin as a loading control. (b) ECV304 cells were transfected with control or EOLA1 shRNA for 24 h and then cultured with or without LPS (100 ng/mL) and the concentration of VCAM-1 in the culture supernatants was determined 6 h after stimulation. Data represent the mean ± SD of three independent experiments ( ∗ p
Figure Legend Snippet: Knockdown of EOLA1 with shRNA inhibits the expression of MT2A and increases the LPS-induced VCAM-1 production in ECV304 cells. (a) ECV304 cells were transfected with control or EOLA1 shRNA for 48 h. The cells were lysed and analyzed by Western blotting with anti-EOLA1 or anti-MT2A antibodies, β -actin as a loading control. (b) ECV304 cells were transfected with control or EOLA1 shRNA for 24 h and then cultured with or without LPS (100 ng/mL) and the concentration of VCAM-1 in the culture supernatants was determined 6 h after stimulation. Data represent the mean ± SD of three independent experiments ( ∗ p

Techniques Used: shRNA, Expressing, Transfection, Western Blot, Cell Culture, Concentration Assay

EOLA1 mediates LPS-induced VCAM-1 production by MT2A. (a) ECV304 cells were transfected with MT2A shRNA or control shRNA, and the cells were lysed and analyzed by Western blotting with anti-MT2A or ant-EOLA1 antibody, β -actin as a loading control. (b) ECV304 was transfected with MT2A shRNA or control shRNA and then stimulated with LPS (100 ng/mL) for 6 h. VCAM-1 production was assayed by ELISA. Data represent the mean ± SD of three independent experiments ( ∗ p
Figure Legend Snippet: EOLA1 mediates LPS-induced VCAM-1 production by MT2A. (a) ECV304 cells were transfected with MT2A shRNA or control shRNA, and the cells were lysed and analyzed by Western blotting with anti-MT2A or ant-EOLA1 antibody, β -actin as a loading control. (b) ECV304 was transfected with MT2A shRNA or control shRNA and then stimulated with LPS (100 ng/mL) for 6 h. VCAM-1 production was assayed by ELISA. Data represent the mean ± SD of three independent experiments ( ∗ p

Techniques Used: Transfection, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay

Subcellular location of EOLA1 is indicated in ECV304 cells. ECV304 cells were transfected with the blank plasmid pEGFP-N2 and the fusion protein expression plasmid pEGFP-EOLA1. After 48 h culture, the transfected cells were fixed with 4% PFA. (a) The images of ECV304 cells were taken under the optical microscope. (b) The cells were incubated with rat anti-GFP antibody (primary antibody) and goat anti-rat antibody (secondary antibody), stained with DAPI, and photographed under LSM510 META laser confocal microscope. (c) The cells were incubated with rabbit anti-EOLA1 polyclonal antibody (primary antibody) and HRP-labeled goat anti-rabbit IgG antibody (secondary antibody), stained with HRP-labeled streptoantibiotin and diaminobenzidine, and embedded with epoxy resin 618. Finally the cells were made into ultrathin sections, stained with lead, and observed and photographed under TECNA110 transmission electron microscope. The white arrow identifies immune sediment deposition in ECV304 cells transfected with pEGFP-EOLA1 by anti-EOLA1 antibody.
Figure Legend Snippet: Subcellular location of EOLA1 is indicated in ECV304 cells. ECV304 cells were transfected with the blank plasmid pEGFP-N2 and the fusion protein expression plasmid pEGFP-EOLA1. After 48 h culture, the transfected cells were fixed with 4% PFA. (a) The images of ECV304 cells were taken under the optical microscope. (b) The cells were incubated with rat anti-GFP antibody (primary antibody) and goat anti-rat antibody (secondary antibody), stained with DAPI, and photographed under LSM510 META laser confocal microscope. (c) The cells were incubated with rabbit anti-EOLA1 polyclonal antibody (primary antibody) and HRP-labeled goat anti-rabbit IgG antibody (secondary antibody), stained with HRP-labeled streptoantibiotin and diaminobenzidine, and embedded with epoxy resin 618. Finally the cells were made into ultrathin sections, stained with lead, and observed and photographed under TECNA110 transmission electron microscope. The white arrow identifies immune sediment deposition in ECV304 cells transfected with pEGFP-EOLA1 by anti-EOLA1 antibody.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Microscopy, Incubation, Staining, Labeling, Transmission Assay

LPS induces the production of EOLA1 and VCAM-1 in ECV304 cells. (a) ECV304 cells were treated with LPS (100 ng/mL) for various periods of time, 0, 3, 6, 12, and 24 h, and the total RNA was analyzed by RT-PCR to EOLA1. (b) The lysates of ECV304 cells were analyzed by Western blotting with antibodies to EOLA1. Data show one representative experiment out of three independent experiments. (c) ECV304 cells were stimulated with LPS (100 ng/mL) for the indicated periods of time and the supernatants were collected and assayed for VCAM-1 production by ELISA. Data represent the mean ± SD of three independent experiments.
Figure Legend Snippet: LPS induces the production of EOLA1 and VCAM-1 in ECV304 cells. (a) ECV304 cells were treated with LPS (100 ng/mL) for various periods of time, 0, 3, 6, 12, and 24 h, and the total RNA was analyzed by RT-PCR to EOLA1. (b) The lysates of ECV304 cells were analyzed by Western blotting with antibodies to EOLA1. Data show one representative experiment out of three independent experiments. (c) ECV304 cells were stimulated with LPS (100 ng/mL) for the indicated periods of time and the supernatants were collected and assayed for VCAM-1 production by ELISA. Data represent the mean ± SD of three independent experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

5) Product Images from "Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation"

Article Title: Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19010279

Diagram of the working principle of the bicistronic reporter system. ( A ) Wildtype bicistronic reporter vector is supposed to transcribe into a bicistronic mRNA possessing 1 cap, 2 ORFs (DsRed and EGFP for the 2 fluorescence wildtype vectors and hluc and hRluc for the 2 luciferase wildtype vectors), and 1 pA tail when transfected into mammalian or insect cells. Translation of the 1st (DsRed or hRluc) and 2nd ORFs (EGFP or hluc) is initiated at the normal 5′ cap and the IRES, respectively; ( B ) Bicistronic construct with 1 pA site may transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the polyadenylation capability of the inserted candidate pA site relative to that of the SV40 pA site; ( C ) Bicistronic construct with 2 pA sites may also transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the relative polyadenylation efficiency of the two inserted candidate pA sites.
Figure Legend Snippet: Diagram of the working principle of the bicistronic reporter system. ( A ) Wildtype bicistronic reporter vector is supposed to transcribe into a bicistronic mRNA possessing 1 cap, 2 ORFs (DsRed and EGFP for the 2 fluorescence wildtype vectors and hluc and hRluc for the 2 luciferase wildtype vectors), and 1 pA tail when transfected into mammalian or insect cells. Translation of the 1st (DsRed or hRluc) and 2nd ORFs (EGFP or hluc) is initiated at the normal 5′ cap and the IRES, respectively; ( B ) Bicistronic construct with 1 pA site may transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the polyadenylation capability of the inserted candidate pA site relative to that of the SV40 pA site; ( C ) Bicistronic construct with 2 pA sites may also transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the relative polyadenylation efficiency of the two inserted candidate pA sites.

Techniques Used: Plasmid Preparation, Fluorescence, Luciferase, Transfection, Construct

6) Product Images from "A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿"

Article Title: A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05791-11

Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter
Figure Legend Snippet: Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter

Techniques Used: Marker, Plasmid Preparation, Mutagenesis

7) Product Images from "Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation"

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation

Journal: Molecular and Cellular Neurosciences

doi: 10.1016/j.mcn.2013.04.006

BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p
Figure Legend Snippet: BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p

Techniques Used: Cell Culture, Expressing

Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p
Figure Legend Snippet: Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p

Techniques Used: Expressing, In Situ Hybridization, Recombinant

8) Product Images from "Codon choice directs constitutive mRNA levels in trypanosomes"

Article Title: Codon choice directs constitutive mRNA levels in trypanosomes

Journal: eLife

doi: 10.7554/eLife.32467

Comparison of GFP expression measured as fluorescence by flow cytometry and protein by western blotting. ( A ) Western blot of total cell lysates from cell lines expressing four different GFP transgenes and a standard curve of recombinant GFP with an N-terminal hexahistidine tag for purification. For cell lines expressing GFPs 065, 226 and 102, 2 × 10 6 cell equivalents were loading in each track and three independent clones of each cell line were used. For the cell line expressing eGFP, the loading was titred from 2 × 10 6 to 0.25 × 10 6 cell equivalents. The amount of recombinant eGFP loaded is indicated. The blot was probed with anti-GFP, then a peroxidase-conjugated donkey anti-rabbit IgG secondary and signal detected by chemiluminescence with x-ray film. The GFP fluorescence of each cell line was measured and the value is shown in arbitrary units. See supplementary file 2 for GFP sequences. ( B ) Plot of quantitation of western blot against fluorescence. The western blot was quantified using ImageJ to analyse x-ray film images and then the standard curve for quantitation. The Pearson’s correlation coefficient between fluorescence and the western blot GFP estimates was calculated and r 2 = 0.965.
Figure Legend Snippet: Comparison of GFP expression measured as fluorescence by flow cytometry and protein by western blotting. ( A ) Western blot of total cell lysates from cell lines expressing four different GFP transgenes and a standard curve of recombinant GFP with an N-terminal hexahistidine tag for purification. For cell lines expressing GFPs 065, 226 and 102, 2 × 10 6 cell equivalents were loading in each track and three independent clones of each cell line were used. For the cell line expressing eGFP, the loading was titred from 2 × 10 6 to 0.25 × 10 6 cell equivalents. The amount of recombinant eGFP loaded is indicated. The blot was probed with anti-GFP, then a peroxidase-conjugated donkey anti-rabbit IgG secondary and signal detected by chemiluminescence with x-ray film. The GFP fluorescence of each cell line was measured and the value is shown in arbitrary units. See supplementary file 2 for GFP sequences. ( B ) Plot of quantitation of western blot against fluorescence. The western blot was quantified using ImageJ to analyse x-ray film images and then the standard curve for quantitation. The Pearson’s correlation coefficient between fluorescence and the western blot GFP estimates was calculated and r 2 = 0.965.

Techniques Used: Expressing, Fluorescence, Flow Cytometry, Cytometry, Western Blot, Recombinant, Purification, Clone Assay, Quantitation Assay

Diagram showing GFP fluorescence detected by flow cytometry in 20 000 cells for each transgene expressing cell line and the parental T. brucei Lister 427 KG procyclic form cell line. Starting with p3605 which directed the expression of MS2bp-GFP-nls with α-tubulin 5’UTR and actin 3’UTR the following changes were made: p3827, substituted β-tubulin 5’UTR; p3854, substituted synthetic 5’UTR (based on pNS11 from [ Siegel et al., 2005 ]); p3866, as p3854 but trans -slice acceptor site mutated from AG to AA; p3943, as p3866 but sequence around initiation codon changed from CCGCCGCCATG to TTTTTTTATG p3965, as p3943 but substituted RAB28 3’UTR p3998, as p3605 but with the ORF altered to contain codons enriched in genes with low expression levels. All sequences are in Supplementary file 1 and the effect of the use of infrequent codons can be seen by comparing p3605 and p3998, in red.
Figure Legend Snippet: Diagram showing GFP fluorescence detected by flow cytometry in 20 000 cells for each transgene expressing cell line and the parental T. brucei Lister 427 KG procyclic form cell line. Starting with p3605 which directed the expression of MS2bp-GFP-nls with α-tubulin 5’UTR and actin 3’UTR the following changes were made: p3827, substituted β-tubulin 5’UTR; p3854, substituted synthetic 5’UTR (based on pNS11 from [ Siegel et al., 2005 ]); p3866, as p3854 but trans -slice acceptor site mutated from AG to AA; p3943, as p3866 but sequence around initiation codon changed from CCGCCGCCATG to TTTTTTTATG p3965, as p3943 but substituted RAB28 3’UTR p3998, as p3605 but with the ORF altered to contain codons enriched in genes with low expression levels. All sequences are in Supplementary file 1 and the effect of the use of infrequent codons can be seen by comparing p3605 and p3998, in red.

Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Expressing, Sequencing

Blocking translation stabilises mRNAs with low geCAI scores more than mRNAs with high geCAI scores. ( A ) Northern blots to measure GFP mRNAs in cell lines expressing GFP with geCAI scores with or without a hairpin structure in the 5’UTR that blocked translation. Three independent clones for each transgene were analysed and GFP; mRNA was quantified by phosphorimaging and values adjusted for loading using rRNA. ( B ) Graph showing the fold increase in mRNA steady state level due to the inclusion of the hairpin in the 5’UTR plotted against the geCAI score for each ORF.
Figure Legend Snippet: Blocking translation stabilises mRNAs with low geCAI scores more than mRNAs with high geCAI scores. ( A ) Northern blots to measure GFP mRNAs in cell lines expressing GFP with geCAI scores with or without a hairpin structure in the 5’UTR that blocked translation. Three independent clones for each transgene were analysed and GFP; mRNA was quantified by phosphorimaging and values adjusted for loading using rRNA. ( B ) Graph showing the fold increase in mRNA steady state level due to the inclusion of the hairpin in the 5’UTR plotted against the geCAI score for each ORF.

Techniques Used: Blocking Assay, Northern Blot, Expressing, Clone Assay

9) Product Images from "A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins"

Article Title: A Second CRM1-Dependent Nuclear Export Signal in the Influenza A Virus NS2 Protein Contributes to the Nuclear Export of Viral Ribonucleoproteins

Journal: Journal of Virology

doi: 10.1128/JVI.06519-11

Characterization of the interaction between influenza virus A/chicken/Helan/12/2004 (H5N1) NEP or its mutants and the nuclear export receptor hCRM1. (A and D) Schematic representation of NEP- and NES-deleted mutants or point mutants used in the mammalian
Figure Legend Snippet: Characterization of the interaction between influenza virus A/chicken/Helan/12/2004 (H5N1) NEP or its mutants and the nuclear export receptor hCRM1. (A and D) Schematic representation of NEP- and NES-deleted mutants or point mutants used in the mammalian

Techniques Used:

Mapping of subcellular localization signals of the influenza virus A/chicken/Helan/12/2004 (H5N1) NEP ΔNES1 . (A) Schematic illustration of influenza A virus NEP ΔNES1 and its truncated derivatives. HeLa cells were transfected with EGFP-NEP
Figure Legend Snippet: Mapping of subcellular localization signals of the influenza virus A/chicken/Helan/12/2004 (H5N1) NEP ΔNES1 . (A) Schematic illustration of influenza A virus NEP ΔNES1 and its truncated derivatives. HeLa cells were transfected with EGFP-NEP

Techniques Used: Transfection

Subcellular localization of transiently expressed influenza virus A/chicken/Helan/12/2004 (H5N1) NEP and its deletion mutant, NEP ΔNES1 , in 293T cells. (A) EGFP-NEP expressed in 293T cells. (B) EGFP-NEP ΔNES1 expressed in 293T cells. Cells
Figure Legend Snippet: Subcellular localization of transiently expressed influenza virus A/chicken/Helan/12/2004 (H5N1) NEP and its deletion mutant, NEP ΔNES1 , in 293T cells. (A) EGFP-NEP expressed in 293T cells. (B) EGFP-NEP ΔNES1 expressed in 293T cells. Cells

Techniques Used: Mutagenesis

Identification of a functional and transferable leucine-rich NES in the influenza virus A/chicken/Helan/12/2004 (H5N1) NEP. (A) Mapping regions in amino acid sequence positions 22 to 45 of the influenza A virus NEP can mediate the nuclear export of two
Figure Legend Snippet: Identification of a functional and transferable leucine-rich NES in the influenza virus A/chicken/Helan/12/2004 (H5N1) NEP. (A) Mapping regions in amino acid sequence positions 22 to 45 of the influenza A virus NEP can mediate the nuclear export of two

Techniques Used: Functional Assay, Sequencing

10) Product Images from "Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus"

Article Title: Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus

Journal: Journal of Virology

doi: 10.1128/JVI.00992-14

Paramyxovirus and filovirus VLPs bud from GSL-enriched lipid microdomains at the plasma membrane. (A) Constructions of HeV M, NiV M, and EBOV VP40 fusion proteins. (B and C) Western blot analysis of eGFP fusion proteins in transfected HEK293T cell lysates
Figure Legend Snippet: Paramyxovirus and filovirus VLPs bud from GSL-enriched lipid microdomains at the plasma membrane. (A) Constructions of HeV M, NiV M, and EBOV VP40 fusion proteins. (B and C) Western blot analysis of eGFP fusion proteins in transfected HEK293T cell lysates

Techniques Used: Western Blot, Transfection

11) Product Images from "A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye"

Article Title: A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028628

Mitochondrial activity responds to a positive or a negative regulator of myogenesis. C2C12 cells were transfected with p53-EGFP, EYFP-JDP2, or EYFP-NLS. Expression of fusion proteins, mitochondria (MitoTracker), and nuclei (Hoechst 33342) were visualized by fluorescence microscopy as described in Methods section. Micrographs were taken at 24 hrs after transfection (cells were in GM for 24 hars) in A, or 72 hrs after transfection (cells were kept in 48 hrs in DM) in B. MitoTracker, Hoechst, and transfected fusion proteins signals are shown in red, blue, and green respectively. White arrow indicates cells expressing an indicated fluorescence fusion protein. Merged image is shown in the middle panel. Intensity of MitoTracker signals were measured using ImageJ program (NIH) (top panel) based on the MitoTracker signal (Bottom panel). Micrographs shown are representative of the samples (n≥6) (scale bar = 10 µm).
Figure Legend Snippet: Mitochondrial activity responds to a positive or a negative regulator of myogenesis. C2C12 cells were transfected with p53-EGFP, EYFP-JDP2, or EYFP-NLS. Expression of fusion proteins, mitochondria (MitoTracker), and nuclei (Hoechst 33342) were visualized by fluorescence microscopy as described in Methods section. Micrographs were taken at 24 hrs after transfection (cells were in GM for 24 hars) in A, or 72 hrs after transfection (cells were kept in 48 hrs in DM) in B. MitoTracker, Hoechst, and transfected fusion proteins signals are shown in red, blue, and green respectively. White arrow indicates cells expressing an indicated fluorescence fusion protein. Merged image is shown in the middle panel. Intensity of MitoTracker signals were measured using ImageJ program (NIH) (top panel) based on the MitoTracker signal (Bottom panel). Micrographs shown are representative of the samples (n≥6) (scale bar = 10 µm).

Techniques Used: Activity Assay, Transfection, Expressing, Fluorescence, Microscopy

12) Product Images from "Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation"

Article Title: Useful Bicistronic Reporter System for Studying Poly(A) Site-Defining cis Elements and Regulation of Alternative Polyadenylation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19010279

Diagram of the working principle of the bicistronic reporter system. ( A ) Wildtype bicistronic reporter vector is supposed to transcribe into a bicistronic mRNA possessing 1 cap, 2 ORFs (DsRed and EGFP for the 2 fluorescence wildtype vectors and hluc and hRluc for the 2 luciferase wildtype vectors), and 1 pA tail when transfected into mammalian or insect cells. Translation of the 1st (DsRed or hRluc) and 2nd ORFs (EGFP or hluc) is initiated at the normal 5′ cap and the IRES, respectively; ( B ) Bicistronic construct with 1 pA site may transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the polyadenylation capability of the inserted candidate pA site relative to that of the SV40 pA site; ( C ) Bicistronic construct with 2 pA sites may also transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the relative polyadenylation efficiency of the two inserted candidate pA sites.
Figure Legend Snippet: Diagram of the working principle of the bicistronic reporter system. ( A ) Wildtype bicistronic reporter vector is supposed to transcribe into a bicistronic mRNA possessing 1 cap, 2 ORFs (DsRed and EGFP for the 2 fluorescence wildtype vectors and hluc and hRluc for the 2 luciferase wildtype vectors), and 1 pA tail when transfected into mammalian or insect cells. Translation of the 1st (DsRed or hRluc) and 2nd ORFs (EGFP or hluc) is initiated at the normal 5′ cap and the IRES, respectively; ( B ) Bicistronic construct with 1 pA site may transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the polyadenylation capability of the inserted candidate pA site relative to that of the SV40 pA site; ( C ) Bicistronic construct with 2 pA sites may also transcribe into (1) a monocistronic mRNA containing only the 1st ORF; (2) a bicistronic mRNA possessing both ORFs as the wildtype vectors; and (3) both of the above, depending on the relative polyadenylation efficiency of the two inserted candidate pA sites.

Techniques Used: Plasmid Preparation, Fluorescence, Luciferase, Transfection, Construct

13) Product Images from "Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus"

Article Title: Development of a Recombination System for the Generation of Occlusion Positive Genetically Modified Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus

Journal: Viruses

doi: 10.3390/v7041599

Characterization of cloned AgMNPV-GFP. ( a ) Schematic representation of plasmid (pI3-gene X) and viral DNA (AgMNPV-I- Ppo I) used in the study and possible viral genomic recombination products in plasmid-genome cotransfections. Genomes that could arise from uncut AgMNPV-I- Ppo I DNA are also included (SR: Single Recombinant; DR: Double Recombinant; dw and up : downstream and upstream polh ORF sequences where recombination could occur) Positions of PCR primers are indicated. A scheme of a wt AgMNPV is also included in order to clarify the origin of the putative PCR products that are shown in the table. ( b ) Table presenting the sizes of expected PCR amplicons in each possible genomic product (primers are those listed in Table 2 ); ( c ) Agarose gel electrophoretic patterns of PCR products using cloned AgMNPV-GFP (DR) and AgMNPV-I -Ppo I DNAs as templates.
Figure Legend Snippet: Characterization of cloned AgMNPV-GFP. ( a ) Schematic representation of plasmid (pI3-gene X) and viral DNA (AgMNPV-I- Ppo I) used in the study and possible viral genomic recombination products in plasmid-genome cotransfections. Genomes that could arise from uncut AgMNPV-I- Ppo I DNA are also included (SR: Single Recombinant; DR: Double Recombinant; dw and up : downstream and upstream polh ORF sequences where recombination could occur) Positions of PCR primers are indicated. A scheme of a wt AgMNPV is also included in order to clarify the origin of the putative PCR products that are shown in the table. ( b ) Table presenting the sizes of expected PCR amplicons in each possible genomic product (primers are those listed in Table 2 ); ( c ) Agarose gel electrophoretic patterns of PCR products using cloned AgMNPV-GFP (DR) and AgMNPV-I -Ppo I DNAs as templates.

Techniques Used: Clone Assay, Plasmid Preparation, Recombinant, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Generation of the parental genome AgMNPV-I -Ppo I. ( a ) Homologous recombination between AgMNPV-2D and pAgI Ppo I substitutes the lacZ ORF for the polyhedrin ORF and provides two I- Ppo I restriction sites. The sequences flanking the polh gene are indicated as up (upstream: 829 bp) and dw (downstream: 883 bp); ( b ) Cell culture dish infected with AgMNPV - I -Ppo I showing lacZ β-galactosidase-positive plaques; ( c ) AgMNPV-I -Ppo I digested with I- Ppo I: release of a 3 kb fragment (see arrowhead) containing the lacZ ORF flanked by I -Ppo I restriction sites; ( d ) Hin dIII restriction patterns of genomic DNA from two AgMNPV-I -Ppo I clones (lanes 1, 2) and wt AgMNPV (lane 3). The Hin dIII G bands are highlighted with boxes; ( e ) Southern blot using a probe targeting downstream polh sequences in band G with (10,194 bp; lanes 1, 2) or without (7262 bp) the lacZ ORF (lane 3).
Figure Legend Snippet: Generation of the parental genome AgMNPV-I -Ppo I. ( a ) Homologous recombination between AgMNPV-2D and pAgI Ppo I substitutes the lacZ ORF for the polyhedrin ORF and provides two I- Ppo I restriction sites. The sequences flanking the polh gene are indicated as up (upstream: 829 bp) and dw (downstream: 883 bp); ( b ) Cell culture dish infected with AgMNPV - I -Ppo I showing lacZ β-galactosidase-positive plaques; ( c ) AgMNPV-I -Ppo I digested with I- Ppo I: release of a 3 kb fragment (see arrowhead) containing the lacZ ORF flanked by I -Ppo I restriction sites; ( d ) Hin dIII restriction patterns of genomic DNA from two AgMNPV-I -Ppo I clones (lanes 1, 2) and wt AgMNPV (lane 3). The Hin dIII G bands are highlighted with boxes; ( e ) Southern blot using a probe targeting downstream polh sequences in band G with (10,194 bp; lanes 1, 2) or without (7262 bp) the lacZ ORF (lane 3).

Techniques Used: Homologous Recombination, Cell Culture, Infection, Clone Assay, Southern Blot

Schematic representation of the recombination system. Parental genome (AgMNPV-I -Ppo I) is linearized by digestion with I -Ppo I, and co-transfected with the transfer vector (pI3). Homologous recombination restores genome viability (re-circularization) generating recombinant progeny. Sequences flanking the original polh gene, where recombination can occur, are indicated as dw (511 bp downstream polh ORF stop codon; AgMNPV genome nucleotide positions 132,239–131,729) and up (610 bp upstream the polh promoter; AgMNPV genome nucleotides positions 934–1543). See [ 4 ] for AgMNPV genome nucleotide numbers.
Figure Legend Snippet: Schematic representation of the recombination system. Parental genome (AgMNPV-I -Ppo I) is linearized by digestion with I -Ppo I, and co-transfected with the transfer vector (pI3). Homologous recombination restores genome viability (re-circularization) generating recombinant progeny. Sequences flanking the original polh gene, where recombination can occur, are indicated as dw (511 bp downstream polh ORF stop codon; AgMNPV genome nucleotide positions 132,239–131,729) and up (610 bp upstream the polh promoter; AgMNPV genome nucleotides positions 934–1543). See [ 4 ] for AgMNPV genome nucleotide numbers.

Techniques Used: Transfection, Plasmid Preparation, Homologous Recombination, Recombinant

A. gemmatalis larvae orally infected with AgMNPV-GFP OBs (red arrowheads) and wt AgMNPV OBs (blue arrowheads) exposed to visible light ( a ) and UV light ( b ); tracheolar ( c ) and hemolymph ( d ) cells extracted from A. gemmatalis larvae infected with AgMNPV-GFP OBs. In the inset panel, polyhedra inside the nucleus of an infected cell are highlighted. ( e ) Results of mean time to death and mortality of A. gemmatalis larvae infected with recombinant AgMNPV-GFP and wt AgMNPV ( n = 20 per treatment). The larvae consumed 50 (1 LD 50 ) and 300 OBs (6 LD 50 ) in parallel experiments.
Figure Legend Snippet: A. gemmatalis larvae orally infected with AgMNPV-GFP OBs (red arrowheads) and wt AgMNPV OBs (blue arrowheads) exposed to visible light ( a ) and UV light ( b ); tracheolar ( c ) and hemolymph ( d ) cells extracted from A. gemmatalis larvae infected with AgMNPV-GFP OBs. In the inset panel, polyhedra inside the nucleus of an infected cell are highlighted. ( e ) Results of mean time to death and mortality of A. gemmatalis larvae infected with recombinant AgMNPV-GFP and wt AgMNPV ( n = 20 per treatment). The larvae consumed 50 (1 LD 50 ) and 300 OBs (6 LD 50 ) in parallel experiments.

Techniques Used: Infection, Recombinant

Generation of AgMNPV-GFP. ( a ) GFP (green fluorescent protein) expression in High Five™ cells co-transfected with linearized AgMNPV-I - P po I and pI3-GFP (schematic in Figure 4 a); ( b ) High Five™ cells infected with cloned AgMNPV-GFP. Cells were infected with recombinant virus recovered from the plug of the agarose overlay from a single selected plaque. The differences in the levels of GFP expression of individual cells are related to the low multiplicity of infection.
Figure Legend Snippet: Generation of AgMNPV-GFP. ( a ) GFP (green fluorescent protein) expression in High Five™ cells co-transfected with linearized AgMNPV-I - P po I and pI3-GFP (schematic in Figure 4 a); ( b ) High Five™ cells infected with cloned AgMNPV-GFP. Cells were infected with recombinant virus recovered from the plug of the agarose overlay from a single selected plaque. The differences in the levels of GFP expression of individual cells are related to the low multiplicity of infection.

Techniques Used: Expressing, Transfection, Infection, Clone Assay, Recombinant

14) Product Images from "SclR, a Basic Helix-Loop-Helix Transcription Factor, Regulates Hyphal Morphology and Promotes Sclerotial Formation in Aspergillus oryzae ▿ ▿ †"

Article Title: SclR, a Basic Helix-Loop-Helix Transcription Factor, Regulates Hyphal Morphology and Promotes Sclerotial Formation in Aspergillus oryzae ▿ ▿ †

Journal: Eukaryotic Cell

doi: 10.1128/EC.00013-11

Construction of the sclR overexpression strain. (a) Construction of the plasmid for sclR overexpression. The construction of the plasmid is described in detail in Materials and Methods. The full-length sclR ORF is inserted into the terminus of the amyB promoter. (b) Confirmation of sclR overexpression strain by Southern blot analysis. Genomic DNAs were digested with EcoRI and hybridized with the probe ( sclR ORF). The expected DNA sizes are shown by the arrow. Lane 1, parent strain; lanes 2 to 5, transformants with pamy215. (c) Confirmation of sclR overexpression by semiquantitative RT-PCR analysis. Total RNA was isolated from the strains cultured on CD or starch-containing CD media for 2 days. Negative-control PCR analysis using each mRNA as a template showed no amplified fragments (data not shown).
Figure Legend Snippet: Construction of the sclR overexpression strain. (a) Construction of the plasmid for sclR overexpression. The construction of the plasmid is described in detail in Materials and Methods. The full-length sclR ORF is inserted into the terminus of the amyB promoter. (b) Confirmation of sclR overexpression strain by Southern blot analysis. Genomic DNAs were digested with EcoRI and hybridized with the probe ( sclR ORF). The expected DNA sizes are shown by the arrow. Lane 1, parent strain; lanes 2 to 5, transformants with pamy215. (c) Confirmation of sclR overexpression by semiquantitative RT-PCR analysis. Total RNA was isolated from the strains cultured on CD or starch-containing CD media for 2 days. Negative-control PCR analysis using each mRNA as a template showed no amplified fragments (data not shown).

Techniques Used: Over Expression, Plasmid Preparation, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Cell Culture, Negative Control, Polymerase Chain Reaction, Amplification

15) Product Images from "GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members"

Article Title: GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members

Journal: Journal of Cell Science

doi: 10.1242/jcs.102798

GTF2IRD1 and GTF2IRD2 antagonism in soleus muscles and distribution of GTF2IRD2 transgenic protein in muscle nuclei. ( A–D ) Detail of soleus muscles in whole hind limb transverse sections from two mice carrying both the GTF2IRD1 and the Gtf2ird2
Figure Legend Snippet: GTF2IRD1 and GTF2IRD2 antagonism in soleus muscles and distribution of GTF2IRD2 transgenic protein in muscle nuclei. ( A–D ) Detail of soleus muscles in whole hind limb transverse sections from two mice carrying both the GTF2IRD1 and the Gtf2ird2

Techniques Used: Transgenic Assay, Mouse Assay

Transgenic expression of GTF2IRD2 and TFII-Iβ in muscle tissue causes fiber type shifts in opposing directions. ( A ) Whole sections through the lower hind limbs of wild-type (WT), TFII-Iβ (TFII-I), GTF2IRD2 (IRD2) and GTF2IRD1 (IRD1) transgenic
Figure Legend Snippet: Transgenic expression of GTF2IRD2 and TFII-Iβ in muscle tissue causes fiber type shifts in opposing directions. ( A ) Whole sections through the lower hind limbs of wild-type (WT), TFII-Iβ (TFII-I), GTF2IRD2 (IRD2) and GTF2IRD1 (IRD1) transgenic

Techniques Used: Transgenic Assay, Expressing

GTF2IRD2 interacts with itself, TFII-I and GTF2IRD1 in decreasing order of affinity. ( A ) Western blots of protein extracts from HEK293 cells transfected with combinations of constructs encoding GFP-tagged and Myc-tagged recombinant proteins before (INPUT)
Figure Legend Snippet: GTF2IRD2 interacts with itself, TFII-I and GTF2IRD1 in decreasing order of affinity. ( A ) Western blots of protein extracts from HEK293 cells transfected with combinations of constructs encoding GFP-tagged and Myc-tagged recombinant proteins before (INPUT)

Techniques Used: Western Blot, Transfection, Construct, Recombinant

GTF2IRD2 localizes peripherally in the nucleus and influences the subnuclear distribution of GTF2IRD1 and TFII-Iβ. ( A ) A series of confocal z-sections through the same nucleus showing the peripheral distribution of GFP-GTF2IRD2. ( B ) Nucleus showing
Figure Legend Snippet: GTF2IRD2 localizes peripherally in the nucleus and influences the subnuclear distribution of GTF2IRD1 and TFII-Iβ. ( A ) A series of confocal z-sections through the same nucleus showing the peripheral distribution of GFP-GTF2IRD2. ( B ) Nucleus showing

Techniques Used:

16) Product Images from "Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity"

Article Title: Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks344

Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.
Figure Legend Snippet: Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.

Techniques Used: Activity Assay, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Expressing, Mutagenesis

Stable RNA secondary structures between a PTC and a downstream exon–exon junction operate as cis -acting elements that increase the levels of the potential NMD substrates. ( A ) Diagram of the reporter mRNAs with different 3′-UTRs. The arrowheads represent the primer pairs used in real-time RT–PCR. ( B ) The steady-state levels of reporter mRNAs expressed in stably transfected polyclonal HEK293 cells as measured by the real-time RT–PCR. The data, expressed as mean ± SD ( n = 3), represent percentages relative to EGFP–NTC. ( C ) The result of the SEAP-reporter analysis using culture supernatants of transfected 293T cells. The data, expressed as mean ± SD ( n = 2), represent relative luminescence units. ( D ) The result of RNA immunoprecipitation from the lysate of HEK293 cells transiently expressing FLAG-Y14. The data, expressed as mean ± SD ( n = 3), represent the ratios (IP pellet/post-IP supernatant) of the mRNA levels. Statistical analyses were done with unpaired two-tailed Student’s t -test (** P
Figure Legend Snippet: Stable RNA secondary structures between a PTC and a downstream exon–exon junction operate as cis -acting elements that increase the levels of the potential NMD substrates. ( A ) Diagram of the reporter mRNAs with different 3′-UTRs. The arrowheads represent the primer pairs used in real-time RT–PCR. ( B ) The steady-state levels of reporter mRNAs expressed in stably transfected polyclonal HEK293 cells as measured by the real-time RT–PCR. The data, expressed as mean ± SD ( n = 3), represent percentages relative to EGFP–NTC. ( C ) The result of the SEAP-reporter analysis using culture supernatants of transfected 293T cells. The data, expressed as mean ± SD ( n = 2), represent relative luminescence units. ( D ) The result of RNA immunoprecipitation from the lysate of HEK293 cells transiently expressing FLAG-Y14. The data, expressed as mean ± SD ( n = 3), represent the ratios (IP pellet/post-IP supernatant) of the mRNA levels. Statistical analyses were done with unpaired two-tailed Student’s t -test (** P

Techniques Used: Quantitative RT-PCR, Stable Transfection, Transfection, Immunoprecipitation, Expressing, Two Tailed Test

17) Product Images from "Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity"

Article Title: Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks344

Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.
Figure Legend Snippet: Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.

Techniques Used: Activity Assay, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Expressing, Mutagenesis

18) Product Images from "Rescue of recombinant peste des petits ruminants virus: creation of a GFP-expressing virus and application in rapid virus neutralization test"

Article Title: Rescue of recombinant peste des petits ruminants virus: creation of a GFP-expressing virus and application in rapid virus neutralization test

Journal: Veterinary Research

doi: 10.1186/1297-9716-43-48

Construction of plasmids for PPRV rescue. ( A ) The cDNA fragments F1, F2, F3 and F4 were reverse transcribed and amplified from PPRV/N75/1 genomic RNA. The hammerhead ribozyme sequence (HamRz) and the hepatitis delta virus ribozyme sequence (HdvRz) were introduced to the 5′ end of F1 and the 3′ end of F4, respectively. All fragments were then subcloned stepwise into the pCI vector to produce plasmid pN75/1. ( B ) DNA fragments Fa (from the HamRz to the GS sequence of M with a Not I site introduced at 3′ end) and Fb (from GE of P gene to the end of F1 with Not I and Pme I sites introduced at the 5′ end) were PCR-amplified from pN75/1 and ligated together to get fragment Fab, then section F1 of pN75/1 was replaced with Fab to get plasmid pN75/1-insertion. The net result was equal to insertion of a morbillivirus gene start (GS) sequence, Not I and Pme I sites, gene end (GE) sequence and CTT intergenic trinucleotide into pN75/1 between nt 3405 and 3406 of the PPRV/N75/1 genome cDNA sequence. ( C ) The GFP ORF with a Kozak sequence at the 5′ end of the ORF was inserted into plasmid pN75/1-insertion to produce plasmid pN75/1-GFP.
Figure Legend Snippet: Construction of plasmids for PPRV rescue. ( A ) The cDNA fragments F1, F2, F3 and F4 were reverse transcribed and amplified from PPRV/N75/1 genomic RNA. The hammerhead ribozyme sequence (HamRz) and the hepatitis delta virus ribozyme sequence (HdvRz) were introduced to the 5′ end of F1 and the 3′ end of F4, respectively. All fragments were then subcloned stepwise into the pCI vector to produce plasmid pN75/1. ( B ) DNA fragments Fa (from the HamRz to the GS sequence of M with a Not I site introduced at 3′ end) and Fb (from GE of P gene to the end of F1 with Not I and Pme I sites introduced at the 5′ end) were PCR-amplified from pN75/1 and ligated together to get fragment Fab, then section F1 of pN75/1 was replaced with Fab to get plasmid pN75/1-insertion. The net result was equal to insertion of a morbillivirus gene start (GS) sequence, Not I and Pme I sites, gene end (GE) sequence and CTT intergenic trinucleotide into pN75/1 between nt 3405 and 3406 of the PPRV/N75/1 genome cDNA sequence. ( C ) The GFP ORF with a Kozak sequence at the 5′ end of the ORF was inserted into plasmid pN75/1-insertion to produce plasmid pN75/1-GFP.

Techniques Used: Amplification, Sequencing, Plasmid Preparation, Polymerase Chain Reaction

19) Product Images from "EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells"

Article Title: EOLA1 Inhibits Lipopolysaccharide-Induced Vascular Cell Adhesion Molecule-1 Expression by Association with MT2A in ECV304 Cells

Journal: International Journal of Inflammation

doi: 10.1155/2015/301562

LPS induces the production of EOLA1 and VCAM-1 in ECV304 cells. (a) ECV304 cells were treated with LPS (100 ng/mL) for various periods of time, 0, 3, 6, 12, and 24 h, and the total RNA was analyzed by RT-PCR to EOLA1. (b) The lysates of ECV304 cells were analyzed by Western blotting with antibodies to EOLA1. Data show one representative experiment out of three independent experiments. (c) ECV304 cells were stimulated with LPS (100 ng/mL) for the indicated periods of time and the supernatants were collected and assayed for VCAM-1 production by ELISA. Data represent the mean ± SD of three independent experiments.
Figure Legend Snippet: LPS induces the production of EOLA1 and VCAM-1 in ECV304 cells. (a) ECV304 cells were treated with LPS (100 ng/mL) for various periods of time, 0, 3, 6, 12, and 24 h, and the total RNA was analyzed by RT-PCR to EOLA1. (b) The lysates of ECV304 cells were analyzed by Western blotting with antibodies to EOLA1. Data show one representative experiment out of three independent experiments. (c) ECV304 cells were stimulated with LPS (100 ng/mL) for the indicated periods of time and the supernatants were collected and assayed for VCAM-1 production by ELISA. Data represent the mean ± SD of three independent experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

20) Product Images from "A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿"

Article Title: A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05791-11

Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter
Figure Legend Snippet: Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter

Techniques Used: Marker, Plasmid Preparation, Mutagenesis

21) Product Images from "DEC1 is a downstream target of TGF-? with sequence-specific transcriptional repressor activities"

Article Title: DEC1 is a downstream target of TGF-? with sequence-specific transcriptional repressor activities

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

doi: 10.1073/pnas.261714999

Targeted disruption of DEC1 gene. ( A ) The wild-type (wt) human DEC1 gene locus is aligned with the targeting vectors. Solid boxes represent exons 1–5. The neomycin cassette is shown as shaded boxes, which replaced exon 4 during targeting. LoxP sites are shown as solid triangles. Cre denotes Cre recombinase. ( B ) Genotyping by PCR was performed with genomic DNA extracted from parental HaCaT (+/+), four heterozygote (+/−), and three DEC1-null (−/−) clones. The positions and orientations of primers used are shown by arrows. The sizes of PCR products are noted on the right.
Figure Legend Snippet: Targeted disruption of DEC1 gene. ( A ) The wild-type (wt) human DEC1 gene locus is aligned with the targeting vectors. Solid boxes represent exons 1–5. The neomycin cassette is shown as shaded boxes, which replaced exon 4 during targeting. LoxP sites are shown as solid triangles. Cre denotes Cre recombinase. ( B ) Genotyping by PCR was performed with genomic DNA extracted from parental HaCaT (+/+), four heterozygote (+/−), and three DEC1-null (−/−) clones. The positions and orientations of primers used are shown by arrows. The sizes of PCR products are noted on the right.

Techniques Used: Polymerase Chain Reaction, Clone Assay

22) Product Images from "A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿"

Article Title: A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures ▿

Journal: Journal of Virology

doi: 10.1128/JVI.05791-11

Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter
Figure Legend Snippet: Marker rescue/marker transfer of the VP23 alanine substitution mutants into the virus. Plasmid pKKBX (KpnI-BstX1) was the backbone for generation of a double mutant in the UL18 and UL19 genes. The deletion (filled box) was replaced with a CMV-eGFP reporter

Techniques Used: Marker, Plasmid Preparation, Mutagenesis

23) Product Images from "Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation"

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation

Journal: Molecular and Cellular Neurosciences

doi: 10.1016/j.mcn.2013.04.006

Wnt2 overexpression increases dendritic protrusion density and influences spine shape on cortical neurons. (A) Representative dendritic segments of cortical neurons expressing either EV or Wnt2. (B) Quantification of dendritic protrusion density. (C) Percent of all dendritic protrusions classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p
Figure Legend Snippet: Wnt2 overexpression increases dendritic protrusion density and influences spine shape on cortical neurons. (A) Representative dendritic segments of cortical neurons expressing either EV or Wnt2. (B) Quantification of dendritic protrusion density. (C) Percent of all dendritic protrusions classified as either spines or filopodia. Quantification of average protrusion length (D) and average spine head width (E) for each treatment. *** p

Techniques Used: Over Expression, Expressing

Expression of Wnt inhibitors or Wnt2 shifts the distribution of dendritic protrusion lengths. (A) Relative frequency distributions of dendritic protrusion length comparing Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ expressing neurons to EV control. (B) Relative frequency distribution of protrusion length comparing Wnt2 expressing neurons to EV control. n = number of neurons: EV(A) n = 31, Wif1 n = 34, Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25; EV(B) n = 29, Wnt2 n = 25. *** p
Figure Legend Snippet: Expression of Wnt inhibitors or Wnt2 shifts the distribution of dendritic protrusion lengths. (A) Relative frequency distributions of dendritic protrusion length comparing Wif1, Sfrp1, Fzd8CRD and Dvl1ΔPDZ expressing neurons to EV control. (B) Relative frequency distribution of protrusion length comparing Wnt2 expressing neurons to EV control. n = number of neurons: EV(A) n = 31, Wif1 n = 34, Sfrp1 n = 23, Fzd8CRD n = 25, Dvl1ΔPDZ n = 25; EV(B) n = 29, Wnt2 n = 25. *** p

Techniques Used: Expressing

Wnt2 overexpression is sufficient to increase cortical dendrite length. (A) Representative cortical neurons expressing either EV or Wnt2. Quantification of the total dendrite length per neuron (B) and the number of dendritic endpoints per neuron (C) for each treatment. (D) Sholl analysis of dendritic complexity comparing Wnt2 expressing neurons to control. (E) Quantification of the number of primary dendrites per neuron for each treatment. * p
Figure Legend Snippet: Wnt2 overexpression is sufficient to increase cortical dendrite length. (A) Representative cortical neurons expressing either EV or Wnt2. Quantification of the total dendrite length per neuron (B) and the number of dendritic endpoints per neuron (C) for each treatment. (D) Sholl analysis of dendritic complexity comparing Wnt2 expressing neurons to control. (E) Quantification of the number of primary dendrites per neuron for each treatment. * p

Techniques Used: Over Expression, Expressing

BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p
Figure Legend Snippet: BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p

Techniques Used: Cell Culture, Expressing

Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p
Figure Legend Snippet: Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p

Techniques Used: Expressing, In Situ Hybridization, Recombinant

24) Product Images from "Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation "

Article Title: Neurotrophin and Wnt signaling cooperatively regulate dendritic spine formation

Journal: Molecular and Cellular Neurosciences

doi: 10.1016/j.mcn.2013.04.006

BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p
Figure Legend Snippet: BDNF and Wnt2 mRNA abundances rise during culture of cortical neurons. Quantification of (A) BDNF and (B) Wnt2 mRNA abundances in cultured cortical neurons at DIV0, DIV10 and DIV14, and in mouse neocortex at P14, normalized to 18S RNA. All sample means were divided by the mean at DIV0 in order to determine the relative mRNA expression levels. ** p

Techniques Used: Cell Culture, Expressing

Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p
Figure Legend Snippet: Wnt2 is expressed in the developing mouse brain and its expression in cortical cultures is increased by BDNF. In situ hybridization images taken from the Allen Developing Brain Atlas ( Allen Developing Mouse Brain Atlas, 2009 ) showing Wnt2 expression (purple color) in the hippocampus (A), primary motor cortex (B), primary visual cortex (C) and striatum (D) at P14. (E) Quantification of Wnt2 mRNA abundance after treatment with recombinant BDNF, TTX or both, normalized to 18S RNA. ** p

Techniques Used: Expressing, In Situ Hybridization, Recombinant

25) Product Images from "Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities"

Article Title: Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

Journal: Molecular and Cellular Biology

doi:

Yng2 and Esa1 form a complex in vivo. Extracts from JC1 cells expressing GFP-HA (lane 1), GFP-HA and myc-Yng2 (lane 2), GFP-HA-Esa1 (HA-Esa1) (lane 3), or GFP-HA-Esa1 and myc-Yng2 (lane 4) were assayed for expression of myc-tagged proteins by Western blot analysis using anti-myc (mAb 9E10) antibody (top panel). 10 mg of total protein from each extract was immunoprecipitated with anti-HA (monoclonal antibody 12CA5) antibody; half of the immunoprecipitate was probed with anti-HA antibody (middle panel), and half was probed with anti-myc antibody (bottom panel). myc-Yng2 was only precipitated when coexpressed with GFP-HA-Esa1. Plasmids used to express proteins were pADGFPHA and pUAD6 (lane 1), pADGFPHA and pADmyc-Yng2 (lane 2), pADGFPHA-Esa1 and pUAD6 (lane 3), or pADGFPHA-Esa1 and pADmyc-Yng2 (lane 4).
Figure Legend Snippet: Yng2 and Esa1 form a complex in vivo. Extracts from JC1 cells expressing GFP-HA (lane 1), GFP-HA and myc-Yng2 (lane 2), GFP-HA-Esa1 (HA-Esa1) (lane 3), or GFP-HA-Esa1 and myc-Yng2 (lane 4) were assayed for expression of myc-tagged proteins by Western blot analysis using anti-myc (mAb 9E10) antibody (top panel). 10 mg of total protein from each extract was immunoprecipitated with anti-HA (monoclonal antibody 12CA5) antibody; half of the immunoprecipitate was probed with anti-HA antibody (middle panel), and half was probed with anti-myc antibody (bottom panel). myc-Yng2 was only precipitated when coexpressed with GFP-HA-Esa1. Plasmids used to express proteins were pADGFPHA and pUAD6 (lane 1), pADGFPHA and pADmyc-Yng2 (lane 2), pADGFPHA-Esa1 and pUAD6 (lane 3), or pADGFPHA-Esa1 and pADmyc-Yng2 (lane 4).

Techniques Used: In Vivo, Expressing, Western Blot, Immunoprecipitation

Yng1, Yng2, and Pho23 are associated with HAT activities. (A) Extracts from JC1 cells expressing GFP-HA (GFP), HA-Yng2 (Yng2), HA-Ing1 (Ing1), HA-Pho23 (Pho23), HA-Yng1 (Yng1), GFP-HA-Esa1 (Esa1), or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA (12CA5) antibody. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom panels) (see Materials and Methods). The left lanes (GFP, Yng2, Ing1, Pho23, and Yng1) and right lanes (Gcn5 and Esa1) of the HAT assay were run on the same gel, but they were exposed to film for 15 and 3 days, respectively. (B) Extracts from JC1 cells expressing GFP-HA (GFP), or GFP-HA fusions with either Yng2 (Yng2), the N-terminal domain (residues 1 to 222) of Yng2 (ΔPHD), the C-terminal domain (residues 222 to 282) of Yng2 (PHD), or Yng2/1 (Yng2 residues 1 to 222 fused to Yng1 residues 155 to 219) were immunoprecipitated with anti-HA antibody. Immunoprecipitates were examined by a Western blot using anti-HA antibody (top), or HAT assays (bottom). Plasmids used to express proteins were pADGFPHA (GFP), pADHA-Yng2 (Yng2), pADHA-Ing1 (Ing1), pADHA-Pho23 (Pho23), pADHA-Yng1 (Yng1), pADGFPHA-Esa1 (Esa1), and pADGFPHA-Gcn5 (Gcn5) (in panel A) and pADGFPHA (GFP), pADGFPHA-Yng2 (Yng2), pADGFPHA-Yng2ΔPHD (ΔPHD), pADGFPHA-Yng2PHD (PHD), and pADGFPHA-Yng2/1 (Yng2/1) (in panel B). Arrows denote migration of relevant proteins. HC denotes antibody heavy chain. The panel on the lower right shows a Coomassie-stained lane from the HAT gel and the migration of histones H3, H2B, H2A, and H4.
Figure Legend Snippet: Yng1, Yng2, and Pho23 are associated with HAT activities. (A) Extracts from JC1 cells expressing GFP-HA (GFP), HA-Yng2 (Yng2), HA-Ing1 (Ing1), HA-Pho23 (Pho23), HA-Yng1 (Yng1), GFP-HA-Esa1 (Esa1), or GFP-HA-Gcn5 (Gcn5) were immunoprecipitated with anti-HA (12CA5) antibody. Immunoprecipitates were split, and half of each sample was examined by Western blot analysis using anti-HA antibody (top), and half was assayed for HAT activity (bottom panels) (see Materials and Methods). The left lanes (GFP, Yng2, Ing1, Pho23, and Yng1) and right lanes (Gcn5 and Esa1) of the HAT assay were run on the same gel, but they were exposed to film for 15 and 3 days, respectively. (B) Extracts from JC1 cells expressing GFP-HA (GFP), or GFP-HA fusions with either Yng2 (Yng2), the N-terminal domain (residues 1 to 222) of Yng2 (ΔPHD), the C-terminal domain (residues 222 to 282) of Yng2 (PHD), or Yng2/1 (Yng2 residues 1 to 222 fused to Yng1 residues 155 to 219) were immunoprecipitated with anti-HA antibody. Immunoprecipitates were examined by a Western blot using anti-HA antibody (top), or HAT assays (bottom). Plasmids used to express proteins were pADGFPHA (GFP), pADHA-Yng2 (Yng2), pADHA-Ing1 (Ing1), pADHA-Pho23 (Pho23), pADHA-Yng1 (Yng1), pADGFPHA-Esa1 (Esa1), and pADGFPHA-Gcn5 (Gcn5) (in panel A) and pADGFPHA (GFP), pADGFPHA-Yng2 (Yng2), pADGFPHA-Yng2ΔPHD (ΔPHD), pADGFPHA-Yng2PHD (PHD), and pADGFPHA-Yng2/1 (Yng2/1) (in panel B). Arrows denote migration of relevant proteins. HC denotes antibody heavy chain. The panel on the lower right shows a Coomassie-stained lane from the HAT gel and the migration of histones H3, H2B, H2A, and H4.

Techniques Used: HAT Assay, Expressing, Immunoprecipitation, Western Blot, Activity Assay, Migration, Staining

Yng2 is localized in the nucleus. Normal JC1 cells expressing GFP (left panels) or GFP-Yng2 (right panels) were grown in HC medium, harvested, and briefly fixed before being mounted in medium containing DAPI (Materials and Methods). Cells were visualized by differential interference contrast microscopy (DIC [top row]), DNA was visualized using UV optics (DAPI [middle row]), and GFP localization was visualized using fluorescein isothiocyanate optics (GFP [bottom row]). Images in each column are of the same field of cells. Plasmids used to express proteins were pADGFPHA (GFP) and pADGFPHA-Yng2 (GFP-Yng2).
Figure Legend Snippet: Yng2 is localized in the nucleus. Normal JC1 cells expressing GFP (left panels) or GFP-Yng2 (right panels) were grown in HC medium, harvested, and briefly fixed before being mounted in medium containing DAPI (Materials and Methods). Cells were visualized by differential interference contrast microscopy (DIC [top row]), DNA was visualized using UV optics (DAPI [middle row]), and GFP localization was visualized using fluorescein isothiocyanate optics (GFP [bottom row]). Images in each column are of the same field of cells. Plasmids used to express proteins were pADGFPHA (GFP) and pADGFPHA-Yng2 (GFP-Yng2).

Techniques Used: Expressing, Microscopy

26) Product Images from "A novel strategy to dissect endogenous gene transcriptional regulation in live cells"

Article Title: A novel strategy to dissect endogenous gene transcriptional regulation in live cells

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2017.04.092

Establishing a model that monitors endogenous ARID1A gene transcription A. Schematic view of our knockin strategy. In our study, the ORF of EGFP (717 bp) was introduced into the ARID1A exon 2 in frame. The ATG start codon of both ORFs is truncated. There are two alternative splicing products of ARID1A . The ATG start codon labeled in black is for the longer transcript, while the one in blue is for the shorter transcript. The knockin was purposely placed in the middle of the ATG start codon for the shorter transcript to destroy it, as indicated by a cross, to ensure that the reporter gene does not interfere with normal ARID1A gene splicing and to keep the regulation of ARID1A transcription by the endogenous promoter intact. The CRISPR-Cas9 target site is indicated by a red asterisk on exon 2. The red bars flanking the reporter ORF in the donor plasmid represent the 1-kb 5′ and 3′ homologous arms to ARID1A genomic locus. B. EGFP-positive clones without sorting (top panel). Fluorescence imaging demonstrates that the number of false EGFP -positive clones is high when cells are cultured for 1 week following the first round of sorting (middle panel). The red circles highlight false-positive cells. However, every cell among the clones, following the second round of sorting, is EGFP positive (lower panel). Scale bar, 200 μm. C. DNA sequencing chromatograms indicate successful knockin of the EGFP gene at the expected integration site. The stop codon of the EGFP gene is underlined by a red bar.
Figure Legend Snippet: Establishing a model that monitors endogenous ARID1A gene transcription A. Schematic view of our knockin strategy. In our study, the ORF of EGFP (717 bp) was introduced into the ARID1A exon 2 in frame. The ATG start codon of both ORFs is truncated. There are two alternative splicing products of ARID1A . The ATG start codon labeled in black is for the longer transcript, while the one in blue is for the shorter transcript. The knockin was purposely placed in the middle of the ATG start codon for the shorter transcript to destroy it, as indicated by a cross, to ensure that the reporter gene does not interfere with normal ARID1A gene splicing and to keep the regulation of ARID1A transcription by the endogenous promoter intact. The CRISPR-Cas9 target site is indicated by a red asterisk on exon 2. The red bars flanking the reporter ORF in the donor plasmid represent the 1-kb 5′ and 3′ homologous arms to ARID1A genomic locus. B. EGFP-positive clones without sorting (top panel). Fluorescence imaging demonstrates that the number of false EGFP -positive clones is high when cells are cultured for 1 week following the first round of sorting (middle panel). The red circles highlight false-positive cells. However, every cell among the clones, following the second round of sorting, is EGFP positive (lower panel). Scale bar, 200 μm. C. DNA sequencing chromatograms indicate successful knockin of the EGFP gene at the expected integration site. The stop codon of the EGFP gene is underlined by a red bar.

Techniques Used: Knock-In, Labeling, CRISPR, Plasmid Preparation, Clone Assay, Fluorescence, Imaging, Cell Culture, DNA Sequencing

27) Product Images from "Human Cytomegalovirus Inhibits Transcription of the CC Chemokine MCP-1 Gene"

Article Title: Human Cytomegalovirus Inhibits Transcription of the CC Chemokine MCP-1 Gene

Journal: Journal of Virology

doi:

The US28 gene product is not required for the inhibition of MCP-1 mRNA induction. (A) Schematic representation of the US27-US29 region of the HCMV genome, the plasmid construct used to delete the US28 ORF, and probes used to characterize the mutant virus. (B) Southern blot of AD169 and ADsubUS28 viral DNA. Viral DNA was digested with Bam HI, resulting in a 6.0-kb fragment containing the US28 ORF for the wild-type virus and in 3.9- and 2.8-kb fragments for the mutant, due to a Bam HI site within the GFP marker. (C) Northern blot of RNA isolated from cells infected with AD169 or the AdsubUS28 mutant and treated with IL-1β (1 ng/ml). RNA was collected at various times p.i. and 2 h after IL-1β addition. An MCP-1-specific cDNA probe and a cDNA probe for a cellular cPLA2 RNA (control) were used. Lanes: 1, mock infection; 2, IL-1β only; 3, IL-1β and HCMV (AD169), 4 h p.i.; 4, IL-1β and HCMV (AD169), 24 h p.i.; 5, IL-1β and HCMV (AD169), 48 h p.i.; 6, IL-1β and HCMV (ADsubUS28), 4 h p.i.; 7, IL-1β and HCMV (ADsubUS28), 24 h p.i.; 8, IL-1β and HCMV (ADsubUS28), 48 h p.i.
Figure Legend Snippet: The US28 gene product is not required for the inhibition of MCP-1 mRNA induction. (A) Schematic representation of the US27-US29 region of the HCMV genome, the plasmid construct used to delete the US28 ORF, and probes used to characterize the mutant virus. (B) Southern blot of AD169 and ADsubUS28 viral DNA. Viral DNA was digested with Bam HI, resulting in a 6.0-kb fragment containing the US28 ORF for the wild-type virus and in 3.9- and 2.8-kb fragments for the mutant, due to a Bam HI site within the GFP marker. (C) Northern blot of RNA isolated from cells infected with AD169 or the AdsubUS28 mutant and treated with IL-1β (1 ng/ml). RNA was collected at various times p.i. and 2 h after IL-1β addition. An MCP-1-specific cDNA probe and a cDNA probe for a cellular cPLA2 RNA (control) were used. Lanes: 1, mock infection; 2, IL-1β only; 3, IL-1β and HCMV (AD169), 4 h p.i.; 4, IL-1β and HCMV (AD169), 24 h p.i.; 5, IL-1β and HCMV (AD169), 48 h p.i.; 6, IL-1β and HCMV (ADsubUS28), 4 h p.i.; 7, IL-1β and HCMV (ADsubUS28), 24 h p.i.; 8, IL-1β and HCMV (ADsubUS28), 48 h p.i.

Techniques Used: Inhibition, Plasmid Preparation, Construct, Mutagenesis, Southern Blot, Marker, Northern Blot, Isolation, Infection

28) Product Images from "Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1), a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins"

Article Title: Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1), a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins

Journal: BMC Genomics

doi: 10.1186/1471-2164-2-3

Nucleotide sequence of dog BCMP1 cDNA . The aminoacid sequence encoded by the ORF appears above the corresponding DNA sequence. The underlined sequence corresponds to the insert of the original clone C60 (see text). ATTTA motifs appear in bold and the polyadenylation signal is highlighted in blue.
Figure Legend Snippet: Nucleotide sequence of dog BCMP1 cDNA . The aminoacid sequence encoded by the ORF appears above the corresponding DNA sequence. The underlined sequence corresponds to the insert of the original clone C60 (see text). ATTTA motifs appear in bold and the polyadenylation signal is highlighted in blue.

Techniques Used: Sequencing

29) Product Images from "In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication"

Article Title: In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Journal: Virology

doi: 10.1016/j.virol.2004.10.045

GIBV phenotypic revertant had lost the EGFP ORF. The absence of the EGFP ORF was confirmed by RT-PCR and nucleotide sequencing of the region corresponding to nucleotides 25,162 to 25,613 of the standard cell adapted IBV. Compared to GIBV, the GIBV revertant produced a smaller RT-PCR product, smaller than the E amplicon, suggesting that the revertant had lost the EGFP and 5a ORFs. (A) RT-PCR and PCR products of the region corresponding to nucleotides 25,162 to 25,613 of GIBV, the phenotypic 5aGFP deletion revertant and the control, E amplicon. The M lanes represent the DNA molecular size marker; lane 1, RT-PCR product of GIBV expressing EGFP; lane 2, the RT-PCR product of the phenotypic revertant that had lost expression of EGFP, and 3, as a control, the PCR product from the E fragment. (B) Alignment of the nucleotide sequences of the region spanning the 5a/EGFP ORF of Δ5aGFP viruses identified four distinct types of EGFP deletions. Italicized nucleotides and the positions starred below the sequence alignment represent conserved sequences. Every mutant lost the EGFP start codon but maintained the 5b start codon (underlined).
Figure Legend Snippet: GIBV phenotypic revertant had lost the EGFP ORF. The absence of the EGFP ORF was confirmed by RT-PCR and nucleotide sequencing of the region corresponding to nucleotides 25,162 to 25,613 of the standard cell adapted IBV. Compared to GIBV, the GIBV revertant produced a smaller RT-PCR product, smaller than the E amplicon, suggesting that the revertant had lost the EGFP and 5a ORFs. (A) RT-PCR and PCR products of the region corresponding to nucleotides 25,162 to 25,613 of GIBV, the phenotypic 5aGFP deletion revertant and the control, E amplicon. The M lanes represent the DNA molecular size marker; lane 1, RT-PCR product of GIBV expressing EGFP; lane 2, the RT-PCR product of the phenotypic revertant that had lost expression of EGFP, and 3, as a control, the PCR product from the E fragment. (B) Alignment of the nucleotide sequences of the region spanning the 5a/EGFP ORF of Δ5aGFP viruses identified four distinct types of EGFP deletions. Italicized nucleotides and the positions starred below the sequence alignment represent conserved sequences. Every mutant lost the EGFP start codon but maintained the 5b start codon (underlined).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Produced, Amplification, Polymerase Chain Reaction, Marker, Expressing, Mutagenesis

Construction of EGFP/Δ5a amplicon. (A) The 5a ORF was replaced with the EGFP ORF. Open boxes represent ORF of the IBV genome. Black lines represent PCR products. Arrows indicate the restriction enzyme sites inserted into the PCR products. (B) PCR products containing EGFP in place of ORF 5a. M represents the 1-kb DNA molecular size marker; lane 1, PCR product of EGFP, denoted as F in (A); lane 2, PCR product of M ORF and junction of 5′ of ORF 5a, denoted as G in (A); lane 3; PCR product encompassing 3′ end of 5a ORF and downstream of the ORF through 3′ UTR of IBV, denoted as H in (A). (C) Underlined are IBV sequences and bold are EGFP sequences.
Figure Legend Snippet: Construction of EGFP/Δ5a amplicon. (A) The 5a ORF was replaced with the EGFP ORF. Open boxes represent ORF of the IBV genome. Black lines represent PCR products. Arrows indicate the restriction enzyme sites inserted into the PCR products. (B) PCR products containing EGFP in place of ORF 5a. M represents the 1-kb DNA molecular size marker; lane 1, PCR product of EGFP, denoted as F in (A); lane 2, PCR product of M ORF and junction of 5′ of ORF 5a, denoted as G in (A); lane 3; PCR product encompassing 3′ end of 5a ORF and downstream of the ORF through 3′ UTR of IBV, denoted as H in (A). (C) Underlined are IBV sequences and bold are EGFP sequences.

Techniques Used: Amplification, Polymerase Chain Reaction, Marker

30) Product Images from "Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing"

Article Title: Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing

Journal: Scientific Reports

doi: 10.1038/srep38277

Melanosome transfer is mediated by plasma membrane vesicles. ( a ) A melanocyte dendrite at E12. Lower panels show magnified images of a square selected from time-lapse movies by Carl Zeiss LSM5 PASCAL ( Supplementary video 5 ), and surface-rendering images by IMARIS 7.6 (Bitplane). Arrows show a membrane bleb that was eventually released from the dendrite. ( b ) From bleb to vesicle changes of the melanocyte plasma membrane during development. Blebbing cells emerge around E6 onward, followed by appearance of vesicle-releasing melanocytes around E10. 30 melanocytes were assessed for each embryo. The definition was made by morphological criteria of membrane tethered EGFP-expressing melanocytes using high-resolution images obtained by confocal microscopy. We define the vesicle-releasing melanocytes as the cells adjacent to which released EGFP-positive vesicles are observed. Blebbing melanocytes are those that exhibit blebs in the plasma membrane. And the rest of the cells are classified as non-blebbing melanocytes. Data are presented as the mean ± SEM. ( c ) An orthogonal confocal image (Nikon A1R) shows an EGFP-positive membrane vesicle (arrow) incorporated into a keratinocyte, whose shape was visualized by phalloidin staining. ( d ) Among 185 vesicles examined in E12 embryos, a majority was found within keratinocytes. In each fraction of inside and outside keratinocytes, the number of pigment-containing vesicles was smaller than that of vacant vesicles. ( e ) A range of different sizes of membrane vesicles with and without a melanosome. 30 vesicles were assessed for each of three embryos (E12). Data are presented as the mean ± SEM. ( f ) Melanocyte-derived gapEGFP+ membrane vesicles are enriched with phosphatidylserine in the outer lipid bilayer (arrows). Membrane vesicles were isolated from the skin of E12 embryos, and subjected to staining with PSvue 550 (red) (See Material and Methods). Lower panels show magnified images of boxed areas in the upper panels.
Figure Legend Snippet: Melanosome transfer is mediated by plasma membrane vesicles. ( a ) A melanocyte dendrite at E12. Lower panels show magnified images of a square selected from time-lapse movies by Carl Zeiss LSM5 PASCAL ( Supplementary video 5 ), and surface-rendering images by IMARIS 7.6 (Bitplane). Arrows show a membrane bleb that was eventually released from the dendrite. ( b ) From bleb to vesicle changes of the melanocyte plasma membrane during development. Blebbing cells emerge around E6 onward, followed by appearance of vesicle-releasing melanocytes around E10. 30 melanocytes were assessed for each embryo. The definition was made by morphological criteria of membrane tethered EGFP-expressing melanocytes using high-resolution images obtained by confocal microscopy. We define the vesicle-releasing melanocytes as the cells adjacent to which released EGFP-positive vesicles are observed. Blebbing melanocytes are those that exhibit blebs in the plasma membrane. And the rest of the cells are classified as non-blebbing melanocytes. Data are presented as the mean ± SEM. ( c ) An orthogonal confocal image (Nikon A1R) shows an EGFP-positive membrane vesicle (arrow) incorporated into a keratinocyte, whose shape was visualized by phalloidin staining. ( d ) Among 185 vesicles examined in E12 embryos, a majority was found within keratinocytes. In each fraction of inside and outside keratinocytes, the number of pigment-containing vesicles was smaller than that of vacant vesicles. ( e ) A range of different sizes of membrane vesicles with and without a melanosome. 30 vesicles were assessed for each of three embryos (E12). Data are presented as the mean ± SEM. ( f ) Melanocyte-derived gapEGFP+ membrane vesicles are enriched with phosphatidylserine in the outer lipid bilayer (arrows). Membrane vesicles were isolated from the skin of E12 embryos, and subjected to staining with PSvue 550 (red) (See Material and Methods). Lower panels show magnified images of boxed areas in the upper panels.

Techniques Used: Expressing, Confocal Microscopy, Staining, Derivative Assay, Isolation

31) Product Images from "Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing"

Article Title: Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing

Journal: Scientific Reports

doi: 10.1038/srep38277

Rho activity is required for the membrane vesicle formation and skin pigmentation. ( a ) Temporally controlled inhibition of Rho activity using the tet-on system. Doxycycline (Dox) was administered into embryos at E7 and E10 to turn on the DN-RhoA- or C3 genes prior to the stages when blebbing and vesicle release would normally start. Effects by Rho inhibition for respective events were evaluated at the stages indicated. ( b – e ) Rho inhibition resulted in a marked reduction of both membrane blebbing ( b , c ) and release of membrane vesicles (arrows) ( d , e ). The photos in ( b ) are selected images from Supplementary video 6 (control gapEGFP) and Video 7 (C3). ( f ) Experimental procedure for pigmentation assay. pT2A-CAGGS-TetOn3G-IRES-Neo r , pCAGGS-T2TP, and pT2A-TRE-gapEGFP/pT2A-TRE-GAPEGFP-C3 genes were electroporated into the neural tube of Hypeco nera (pigmented strain) at E2. A skin tissue taken from E9 embryo was dissociated into single cells, and gene-electroporated melanocytes were enriched in G418-containing culture medium. Subsequently, an aggregate of enriched melanocytes was transplanted into host embryos of White leghorn (non-pigmented) at E2. The C3 gene turned on by Dox administration at E10. See Material and Methods. ( g ) Melanin particles in 1 mm 2 of the skin were quantified by Nikon software NIS-elements. All values of statistical data are shown as the mean ± SEM. Statistical significance was calculated using Student’s t-test: *P
Figure Legend Snippet: Rho activity is required for the membrane vesicle formation and skin pigmentation. ( a ) Temporally controlled inhibition of Rho activity using the tet-on system. Doxycycline (Dox) was administered into embryos at E7 and E10 to turn on the DN-RhoA- or C3 genes prior to the stages when blebbing and vesicle release would normally start. Effects by Rho inhibition for respective events were evaluated at the stages indicated. ( b – e ) Rho inhibition resulted in a marked reduction of both membrane blebbing ( b , c ) and release of membrane vesicles (arrows) ( d , e ). The photos in ( b ) are selected images from Supplementary video 6 (control gapEGFP) and Video 7 (C3). ( f ) Experimental procedure for pigmentation assay. pT2A-CAGGS-TetOn3G-IRES-Neo r , pCAGGS-T2TP, and pT2A-TRE-gapEGFP/pT2A-TRE-GAPEGFP-C3 genes were electroporated into the neural tube of Hypeco nera (pigmented strain) at E2. A skin tissue taken from E9 embryo was dissociated into single cells, and gene-electroporated melanocytes were enriched in G418-containing culture medium. Subsequently, an aggregate of enriched melanocytes was transplanted into host embryos of White leghorn (non-pigmented) at E2. The C3 gene turned on by Dox administration at E10. See Material and Methods. ( g ) Melanin particles in 1 mm 2 of the skin were quantified by Nikon software NIS-elements. All values of statistical data are shown as the mean ± SEM. Statistical significance was calculated using Student’s t-test: *P

Techniques Used: Activity Assay, Inhibition, Software

32) Product Images from "Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing"

Article Title: Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing

Journal: Scientific Reports

doi: 10.1038/srep38277

Rho activity is required for the membrane vesicle formation and skin pigmentation. ( a ) Temporally controlled inhibition of Rho activity using the tet-on system. Doxycycline (Dox) was administered into embryos at E7 and E10 to turn on the DN-RhoA- or C3 genes prior to the stages when blebbing and vesicle release would normally start. Effects by Rho inhibition for respective events were evaluated at the stages indicated. ( b – e ) Rho inhibition resulted in a marked reduction of both membrane blebbing ( b , c ) and release of membrane vesicles (arrows) ( d , e ). The photos in ( b ) are selected images from Supplementary video 6 (control gapEGFP) and Video 7 (C3). ( f ) Experimental procedure for pigmentation assay. pT2A-CAGGS-TetOn3G-IRES-Neo r , pCAGGS-T2TP, and pT2A-TRE-gapEGFP/pT2A-TRE-GAPEGFP-C3 genes were electroporated into the neural tube of Hypeco nera (pigmented strain) at E2. A skin tissue taken from E9 embryo was dissociated into single cells, and gene-electroporated melanocytes were enriched in G418-containing culture medium. Subsequently, an aggregate of enriched melanocytes was transplanted into host embryos of White leghorn (non-pigmented) at E2. The C3 gene turned on by Dox administration at E10. See Material and Methods. ( g ) Melanin particles in 1 mm 2 of the skin were quantified by Nikon software NIS-elements. All values of statistical data are shown as the mean ± SEM. Statistical significance was calculated using Student’s t-test: *P
Figure Legend Snippet: Rho activity is required for the membrane vesicle formation and skin pigmentation. ( a ) Temporally controlled inhibition of Rho activity using the tet-on system. Doxycycline (Dox) was administered into embryos at E7 and E10 to turn on the DN-RhoA- or C3 genes prior to the stages when blebbing and vesicle release would normally start. Effects by Rho inhibition for respective events were evaluated at the stages indicated. ( b – e ) Rho inhibition resulted in a marked reduction of both membrane blebbing ( b , c ) and release of membrane vesicles (arrows) ( d , e ). The photos in ( b ) are selected images from Supplementary video 6 (control gapEGFP) and Video 7 (C3). ( f ) Experimental procedure for pigmentation assay. pT2A-CAGGS-TetOn3G-IRES-Neo r , pCAGGS-T2TP, and pT2A-TRE-gapEGFP/pT2A-TRE-GAPEGFP-C3 genes were electroporated into the neural tube of Hypeco nera (pigmented strain) at E2. A skin tissue taken from E9 embryo was dissociated into single cells, and gene-electroporated melanocytes were enriched in G418-containing culture medium. Subsequently, an aggregate of enriched melanocytes was transplanted into host embryos of White leghorn (non-pigmented) at E2. The C3 gene turned on by Dox administration at E10. See Material and Methods. ( g ) Melanin particles in 1 mm 2 of the skin were quantified by Nikon software NIS-elements. All values of statistical data are shown as the mean ± SEM. Statistical significance was calculated using Student’s t-test: *P

Techniques Used: Activity Assay, Inhibition, Software

33) Product Images from "The Identification of Trans-acting Factors That Regulate the Expression of GDF5 via the Osteoarthritis Susceptibility SNP rs143383"

Article Title: The Identification of Trans-acting Factors That Regulate the Expression of GDF5 via the Osteoarthritis Susceptibility SNP rs143383

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003557

Proposed binding model of the four trans -acting factors to rs143383. A region (+9 to +301 relative to the transcription start site) of the GDF5 5′UTR is depicted, with the sequence immediately flanking rs143383 (T allele underlined) shown. We propose that DEAF-1 binds directly to rs143383 (at the TTGG site) and that Sp1 and Sp3 bind just upstream (to the Sp site GGGCGG), mediating a repressive effect through DEAF-1. P15 may be interacting with the repressive multi-protein complex and serving as a linker with the general transcription machinery. ORF is the open reading frame of GDF5 whilst ATG is the translation initiation codon.
Figure Legend Snippet: Proposed binding model of the four trans -acting factors to rs143383. A region (+9 to +301 relative to the transcription start site) of the GDF5 5′UTR is depicted, with the sequence immediately flanking rs143383 (T allele underlined) shown. We propose that DEAF-1 binds directly to rs143383 (at the TTGG site) and that Sp1 and Sp3 bind just upstream (to the Sp site GGGCGG), mediating a repressive effect through DEAF-1. P15 may be interacting with the repressive multi-protein complex and serving as a linker with the general transcription machinery. ORF is the open reading frame of GDF5 whilst ATG is the translation initiation codon.

Techniques Used: Binding Assay, Sequencing

34) Product Images from "The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression"

Article Title: The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

Journal: Journal of Virology

doi:

Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.
Figure Legend Snippet: Schematic diagram of recombinant HCMVs containing mutations of the MIE regulatory region. HCMV genome and its unique long (U L ) and short (U S ), internal repeat long (IR L ) and short (IR S ), terminal repeat long (TR L ) and short (TR S ), and a-sequence components are depicted. Locations of MIE regulatory region, MIE gene exons 1 to 4 (open boxes), and putative UL128 gene (open box) within U L component are shown. The MIE regulatory region of the WT is composed of proximal promoter (+1 to −64), enhancer (Enh; -65 to -550), unique region (-551 to -749), and modulator (Mod; -750 to -1140). Numerical base positions are assigned relative to start site of MIE RNAs. Recombinant HCMVs rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , rΔ-640/-1108 SVgfp , and r SVgfp were derived from rΔM SVgpt ). rΔ-300/-640 SVgfp , rΔ-300/-1108 SVgfp , and rΔ-640/-1108 SVgfp have deletions from -300 to -640, -300 to -1108, and -640 to -1108, respectively. An enhancerless SV40 early promoter (−138 to +57), gfp ORF, and SV40 early intron and polyadenylation signal were inserted at the site of deletion. r SVgfp has the same insertion at -640 but has no deletion. rΔM SVgpt has a deletion of -640 to -1108 and insertion of the SV40 early transcription unit containing the gpt ORF.

Techniques Used: Recombinant, Sequencing, Derivative Assay

35) Product Images from "Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus"

Article Title: Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme Nor-1 in Aspergillus parasiticus

Journal: Mycological research

doi: 10.1016/j.mycres.2009.01.013

Restriction endonuclease map of plasmid pAPCGFPNFNB. The 2.4 kb nor-1 promoter/ORF was fused in frame to the 0.7 kb egfp coding region, followed by the 1.8 kb nor-1 terminator. The 7.4 kb niaD fragment was inserted as a selectable marker for transformation
Figure Legend Snippet: Restriction endonuclease map of plasmid pAPCGFPNFNB. The 2.4 kb nor-1 promoter/ORF was fused in frame to the 0.7 kb egfp coding region, followed by the 1.8 kb nor-1 terminator. The 7.4 kb niaD fragment was inserted as a selectable marker for transformation

Techniques Used: Plasmid Preparation, Marker, Transformation Assay

36) Product Images from "Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo"

Article Title: Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007487

cDNA structure and expression of GP33. (A) ]. The ORF predicted initially as GP33 (hatched box; GP33s) and the ORF determined by RACE (closed boxes; GP33) are shown. An open arrowhead indicates the location of the DRY motif. “ins TGAT” is the mutation containing a stop codon and a 1-bp insertion to generate the GP33-defective virus, Δ33. (B) Multiple sequence alignment of GP33 homologs, HCMV UL33, MCMV M33, and RCMV R33, was performed by ClustalW analysis (DDBJ, ver. 2.1) with the default parameters except for GAP OPEN 100, and the obtained bootstrapped tree is depicted by TreeViewX software. (C) Localization of GP33-EGFP fusion protein in COS-7 cells transiently transfected with pEGFP-GP33. (D) Lysates of cells transfected with pcDNA3 empty vector (lane 1) or pcDNA-GP33F (lane 2) were separated on SDS-10% polyacrylamide gel. Proteins in the gel were transferred to polyvinylidene difluoride membrane (Millipore), and reacted with monoclonal anti-FLAG tag antibody followed by peroxidase-conjugated anti-mouse IgG. An arrowhead indicates the expected full-length GP33 band.
Figure Legend Snippet: cDNA structure and expression of GP33. (A) ]. The ORF predicted initially as GP33 (hatched box; GP33s) and the ORF determined by RACE (closed boxes; GP33) are shown. An open arrowhead indicates the location of the DRY motif. “ins TGAT” is the mutation containing a stop codon and a 1-bp insertion to generate the GP33-defective virus, Δ33. (B) Multiple sequence alignment of GP33 homologs, HCMV UL33, MCMV M33, and RCMV R33, was performed by ClustalW analysis (DDBJ, ver. 2.1) with the default parameters except for GAP OPEN 100, and the obtained bootstrapped tree is depicted by TreeViewX software. (C) Localization of GP33-EGFP fusion protein in COS-7 cells transiently transfected with pEGFP-GP33. (D) Lysates of cells transfected with pcDNA3 empty vector (lane 1) or pcDNA-GP33F (lane 2) were separated on SDS-10% polyacrylamide gel. Proteins in the gel were transferred to polyvinylidene difluoride membrane (Millipore), and reacted with monoclonal anti-FLAG tag antibody followed by peroxidase-conjugated anti-mouse IgG. An arrowhead indicates the expected full-length GP33 band.

Techniques Used: Expressing, Mutagenesis, Sequencing, Software, Transfection, Plasmid Preparation, FLAG-tag

37) Product Images from "Regulation of eIF2? Phosphorylation by Different Functions That Act during Discrete Phases in the Herpes Simplex Virus Type 1 Life Cycle"

Article Title: Regulation of eIF2? Phosphorylation by Different Functions That Act during Discrete Phases in the Herpes Simplex Virus Type 1 Life Cycle

Journal: Journal of Virology

doi: 10.1128/JVI.77.20.10917-10928.2003

Structure of the HSV-1 recombinants and the strategy for repairing mutant alleles. (A) The wild-type HSV-1 genome. Boxed regions, inverted terminal repeat (TR) regions that flank the unique short (U S ) and unique long (U L ) components (solid lines). The location and orientation of both copies of the γ 1 34.5 gene are indicated. The U S -TR junction region containing the Us10, Us11, and Us12 ORFs (open rectangles; stars, respective cis -acting promoter elements) appears expanded beneath the genome. Arrows above boxes extending from the promoter element, mRNA transcripts that encode gene products. All of these mRNAs are polyadenylated at a common polyadenylation signal (not depicted) downstream from the Us10 ORF. The Us12 mRNA is spliced, and joining two noncontiguous regions to form the mRNA indicates this. Solid lines below the restriction enzyme sites, probes used to detect the Us10-to-US12 region. (B) Genomic structure of recombinant HSV-1 mutants. The genomes represented are all those of γ 1 34.5 deletion mutants that contain the β-glucuronidase gene at both γ 1 34.5 loci. Δ34.5-(IE)Us11 virus contains an additional 583-bp deletion spanning the U S -TR junction, removing the Us11 promoter along with most of the Us12 ORF, including the initiation codon, andresulting in the production of an IE mRNA that initiates from the Us12 promoter and that encodes the Us11 polypeptide. The ΔUs11 and pAUs11 mutations both insert the EGFP ORF and SV40 polyadenylation signal downstream of the Us11 promoter and Us12 stop codon, such that the Us12 ORF remains intact and the EGFP ORF is expressed from the Us11 promoter. The ΔUs11 mutation inserts the EGFP ORF and SV40 polyadenylation signal in place of the Us11 ORF, resulting in the complete removal of Us11 coding sequences from the virus and the disruption of the Us10 ORF, whereas the pAUs11 mutation, which places the EGFP ORF and SV40 polyadenylation signal upstream of the Us11 ORF, creates a Us11 null allele by polyadenylating transcripts before they reach the Us11 ORF. This eliminates the production of an mRNA capable of encoding the Us11 protein and preserves the integrity of the Us10 ORF and its promoter.
Figure Legend Snippet: Structure of the HSV-1 recombinants and the strategy for repairing mutant alleles. (A) The wild-type HSV-1 genome. Boxed regions, inverted terminal repeat (TR) regions that flank the unique short (U S ) and unique long (U L ) components (solid lines). The location and orientation of both copies of the γ 1 34.5 gene are indicated. The U S -TR junction region containing the Us10, Us11, and Us12 ORFs (open rectangles; stars, respective cis -acting promoter elements) appears expanded beneath the genome. Arrows above boxes extending from the promoter element, mRNA transcripts that encode gene products. All of these mRNAs are polyadenylated at a common polyadenylation signal (not depicted) downstream from the Us10 ORF. The Us12 mRNA is spliced, and joining two noncontiguous regions to form the mRNA indicates this. Solid lines below the restriction enzyme sites, probes used to detect the Us10-to-US12 region. (B) Genomic structure of recombinant HSV-1 mutants. The genomes represented are all those of γ 1 34.5 deletion mutants that contain the β-glucuronidase gene at both γ 1 34.5 loci. Δ34.5-(IE)Us11 virus contains an additional 583-bp deletion spanning the U S -TR junction, removing the Us11 promoter along with most of the Us12 ORF, including the initiation codon, andresulting in the production of an IE mRNA that initiates from the Us12 promoter and that encodes the Us11 polypeptide. The ΔUs11 and pAUs11 mutations both insert the EGFP ORF and SV40 polyadenylation signal downstream of the Us11 promoter and Us12 stop codon, such that the Us12 ORF remains intact and the EGFP ORF is expressed from the Us11 promoter. The ΔUs11 mutation inserts the EGFP ORF and SV40 polyadenylation signal in place of the Us11 ORF, resulting in the complete removal of Us11 coding sequences from the virus and the disruption of the Us10 ORF, whereas the pAUs11 mutation, which places the EGFP ORF and SV40 polyadenylation signal upstream of the Us11 ORF, creates a Us11 null allele by polyadenylating transcripts before they reach the Us11 ORF. This eliminates the production of an mRNA capable of encoding the Us11 protein and preserves the integrity of the Us10 ORF and its promoter.

Techniques Used: Mutagenesis, Recombinant

38) Product Images from "Influence of Host Gene Transcription Level and Orientation on HIV-1 Latency in a Primary-Cell Model ▿"

Article Title: Influence of Host Gene Transcription Level and Orientation on HIV-1 Latency in a Primary-Cell Model ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02536-10

Generation of latently infected primary CD4 + T cells. (A) Experimental approach to generating acutely infected, persistently infected, and latently infected CD4 + T cells. Activated Bcl-2-transduced CD4 + T cells were infected with NL4-3-Δ6-drEGFP.
Figure Legend Snippet: Generation of latently infected primary CD4 + T cells. (A) Experimental approach to generating acutely infected, persistently infected, and latently infected CD4 + T cells. Activated Bcl-2-transduced CD4 + T cells were infected with NL4-3-Δ6-drEGFP.

Techniques Used: Infection

39) Product Images from "Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity"

Article Title: Evidence that the Upf1-related molecular motor scans the 3?-UTR to ensure mRNA integrity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks344

Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.
Figure Legend Snippet: Close proximity between a PTC and a downstream exon–exon junction bypasses the need of the helicase activity of Upf1. ( A ) The left diagram shows reporter mRNAs with different spacer lengths between the EGFP–ORF and the exon–exon junction. Arrowheads represent the primer pairs used in real-time RT–PCR. The right panel indicates the mean fluorescence intensities of EGFP obtained by flow cytometric analysis of the HEK293T cells expressing one of the reporter mRNAs and the wild-type (black bars) or the R844C mutant (gray bars) of Upf1. The data represent mean ± standard error of the mean (SEM) ( n = 3). ( B ) The levels of reporter mRNAs expressed in HeLa cells. The data, expressed as mean ± SEM ( n = 3), represent a fold difference of the mRNA levels compared to that of SD100nt with the wild-type of Upf1.

Techniques Used: Activity Assay, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Expressing, Mutagenesis

40) Product Images from "Molecular, Biological, and In Vivo Characterization of the Guinea Pig Cytomegalovirus (CMV) Homologs of the Human CMV Matrix Proteins pp71 (UL82) and pp65 (UL83)"

Article Title: Molecular, Biological, and In Vivo Characterization of the Guinea Pig Cytomegalovirus (CMV) Homologs of the Human CMV Matrix Proteins pp71 (UL82) and pp65 (UL83)

Journal: Journal of Virology

doi: 10.1128/JVI.78.18.9872-9889.2004

Nuclear localization of GP83 and HCMV pp65-EGFP fusion proteins. (A) Map of expression constructs. The peGFP-C1 and modified EGFP plasmids encoding C-terminal fusions of either GP83 ORF (peGFPGP83) or HCMV pp65 (peGFPUL83) are described in Materials and Methods. Restriction sites utilized for cloning purposes are indicated, as well as putative NLS predicted by coding sequence analysis. (B) Evaluation of fluorescence following transfection. Expression plasmids (5 μg) were transfected onto separate confluent monolayers of a six-well dish, and expression was allowed to proceed for 24 h prior to cell fixation with 4% paraformaldehyde and detection of autofluorescence as described in Materials and Methods. Transfection data are shown for peGFPGP83 (a and b, duplicate samples), peGFPUL83 (c), and the EGFP control plasmid, peGFP-C1 (d).
Figure Legend Snippet: Nuclear localization of GP83 and HCMV pp65-EGFP fusion proteins. (A) Map of expression constructs. The peGFP-C1 and modified EGFP plasmids encoding C-terminal fusions of either GP83 ORF (peGFPGP83) or HCMV pp65 (peGFPUL83) are described in Materials and Methods. Restriction sites utilized for cloning purposes are indicated, as well as putative NLS predicted by coding sequence analysis. (B) Evaluation of fluorescence following transfection. Expression plasmids (5 μg) were transfected onto separate confluent monolayers of a six-well dish, and expression was allowed to proceed for 24 h prior to cell fixation with 4% paraformaldehyde and detection of autofluorescence as described in Materials and Methods. Transfection data are shown for peGFPGP83 (a and b, duplicate samples), peGFPUL83 (c), and the EGFP control plasmid, peGFP-C1 (d).

Techniques Used: Expressing, Construct, Modification, Clone Assay, Sequencing, Fluorescence, Transfection, Plasmid Preparation

Related Articles

Amplification:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Synthesized:

Article Title: Overexpression of Programmed Death Ligands in Naturally Occurring Postweaning Multisystemic Wasting Syndrome
Article Snippet: .. Residual DNA was removed from the total RNA samples, and first-strand cDNA was synthesized from 1 μg of total RNA with a PrimeScript RT Reagent Kit with gDNA eraser (perfect real-time; Takara, code: RR047A) according to the manufacturer's protocol. .. Briefly, residual DNA was removed as follows: 10 μL total volume of 1 μg total RNA, 2 μL 5×gDNA eraser buffer, 1 μL gDNA eraser and RNase-free dH2 O at 42°C for 2 min, and then put on the ice.

Quantitative RT-PCR:

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: .. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. RT-qPCR reactions were run in 48-well reaction plates with an Illumina Eco Real-Time PCR System and a SYBR green kit (TaKaRa, Japan), according to the manufacturer's recommendations.

Real-time Polymerase Chain Reaction:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: .. For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
Article Snippet: .. For Quantitative real-time PCR (RT-PCR), 500 ng of treated RNA was directly reverse transcribed using Prime Script RT Master Mix (Takara, Japan) and either random or oligo(dT) primers. .. Reverse transcription of miRNA was performed using a New Poly(A) Tailing Kit (ThermoFisher Scientific, China). mRNA was reverse transcribed into cDNA with a PrimeScript RT Master Mix Kit (Takara, RR036A, Japan). cDNA was amplified using Universal SYBR Green Master Mix (4,913,914,001, Roche, Shanghai, China).

Article Title: Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay
Article Snippet: .. RT-PCR After isolating total RNA from samples, it was reverse transcribed into cDNA by following the manufacturer's instructions in PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). .. Using Premix Taq™ kit, the PCR assay was performed under the following conditions: 1 μ L each primer (10 μ m), 25 μ L premix, 1 μ L cDNA, and 22 μ L distilled water.

Luciferase:

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Polymerase Chain Reaction:

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
Article Snippet: .. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) Briefly, cDNA copies of total RNA were obtained using PrimerScript RT reagent Kit (TaKaRa, Japan). .. RT-qPCR reactions were run in 48-well reaction plates with an Illumina Eco Real-Time PCR System and a SYBR green kit (TaKaRa, Japan), according to the manufacturer's recommendations.

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

other:

Article Title: HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period
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    TaKaRa egfp open reading frame
    Modulation of rostro-caudal and dorso-ventral axis specification induces the development of PHOX2B -expressing NCCs from hPSCs. ( a ) A diagram of the culture conditions for the modification of rostro-caudal and dorso-ventral axis specification of hPSCs. ( b ) A heat map image showing the percentage of <t>eGFP</t> + cells on day 10 of differentiation using KhES1 PHOX2B::eGFP under various conditions. ( c ) Representative FCM plots of day 10 KhES1 PHOX2B::eGFP-derived aggregates under the indicated conditions (i–iv) of ( b ). ( d ) <t>RT-PCR</t> analyses for PHOX2B , SOX1 , PAX6 , SOX10 , FOXD3 , HOXB1 , HOXB2 , HOXB4 , HOXB6 , HOXB8 and HOXC9 in day 10 aggregates under conditions (i–iv). The right diagram shows the expression pattern of HOX genes in the rhombomere (r1–8) and the spinal cord (cervical and thoracic) region. SB = SB431542, CHIR = CHIR 99021, RA = retinoic acid, Pur = Purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord.
    Egfp Open Reading Frame, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa egfp enhanced green fluorescent protein coding region
    Functional characterization of the signal peptide deletion in <t>COL4A3.</t> Single confocal sections of HEK293 cells expressing <t>EGFP</t> N-terminus fused either with the entire COL4A3 signal peptide (SP-wt-hybEGFP, top panels) or the 8-amino-acid deleted signal peptide (SP-del-hybEGFP, middle panels). Positive control cells, expressing a soluble EGFP (pEGFP-N1) are also shown (bottom panels). DAPI, 4',6-diamidino-2-phenylindole; EGFP, Enhanced green fluorescent protein. Scale bar: 10 μm.
    Egfp Enhanced Green Fluorescent Protein Coding Region, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa egfp orf
    Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. <t>EGFP</t> was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 <t>ORF.</t> The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.
    Egfp Orf, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Modulation of rostro-caudal and dorso-ventral axis specification induces the development of PHOX2B -expressing NCCs from hPSCs. ( a ) A diagram of the culture conditions for the modification of rostro-caudal and dorso-ventral axis specification of hPSCs. ( b ) A heat map image showing the percentage of eGFP + cells on day 10 of differentiation using KhES1 PHOX2B::eGFP under various conditions. ( c ) Representative FCM plots of day 10 KhES1 PHOX2B::eGFP-derived aggregates under the indicated conditions (i–iv) of ( b ). ( d ) RT-PCR analyses for PHOX2B , SOX1 , PAX6 , SOX10 , FOXD3 , HOXB1 , HOXB2 , HOXB4 , HOXB6 , HOXB8 and HOXC9 in day 10 aggregates under conditions (i–iv). The right diagram shows the expression pattern of HOX genes in the rhombomere (r1–8) and the spinal cord (cervical and thoracic) region. SB = SB431542, CHIR = CHIR 99021, RA = retinoic acid, Pur = Purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord.

    Journal: Scientific Reports

    Article Title: Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition

    doi: 10.1038/s41598-018-31256-1

    Figure Lengend Snippet: Modulation of rostro-caudal and dorso-ventral axis specification induces the development of PHOX2B -expressing NCCs from hPSCs. ( a ) A diagram of the culture conditions for the modification of rostro-caudal and dorso-ventral axis specification of hPSCs. ( b ) A heat map image showing the percentage of eGFP + cells on day 10 of differentiation using KhES1 PHOX2B::eGFP under various conditions. ( c ) Representative FCM plots of day 10 KhES1 PHOX2B::eGFP-derived aggregates under the indicated conditions (i–iv) of ( b ). ( d ) RT-PCR analyses for PHOX2B , SOX1 , PAX6 , SOX10 , FOXD3 , HOXB1 , HOXB2 , HOXB4 , HOXB6 , HOXB8 and HOXC9 in day 10 aggregates under conditions (i–iv). The right diagram shows the expression pattern of HOX genes in the rhombomere (r1–8) and the spinal cord (cervical and thoracic) region. SB = SB431542, CHIR = CHIR 99021, RA = retinoic acid, Pur = Purmorphamine, BMP = BMP4, NT = neural tube, NCC = neural crest cell, HB = hindbrain, SC = spinal cord.

    Article Snippet: Then, the 1 kbp 5′ homology arm (PCR amplified), T2A peptide sequence (annealed oligonucleotide pair) and eGFP open reading frame (ORF; without first ATG; PCR amplified) were cloned into the 5′ side of the loxP-neo-loxP cassette vector using the In-Fusion HD cloning kit (Clontech) in a seamless manner.

    Techniques: Expressing, Modification, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

    Functional characterization of the signal peptide deletion in COL4A3. Single confocal sections of HEK293 cells expressing EGFP N-terminus fused either with the entire COL4A3 signal peptide (SP-wt-hybEGFP, top panels) or the 8-amino-acid deleted signal peptide (SP-del-hybEGFP, middle panels). Positive control cells, expressing a soluble EGFP (pEGFP-N1) are also shown (bottom panels). DAPI, 4',6-diamidino-2-phenylindole; EGFP, Enhanced green fluorescent protein. Scale bar: 10 μm.

    Journal: PLoS ONE

    Article Title: Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis

    doi: 10.1371/journal.pone.0178630

    Figure Lengend Snippet: Functional characterization of the signal peptide deletion in COL4A3. Single confocal sections of HEK293 cells expressing EGFP N-terminus fused either with the entire COL4A3 signal peptide (SP-wt-hybEGFP, top panels) or the 8-amino-acid deleted signal peptide (SP-del-hybEGFP, middle panels). Positive control cells, expressing a soluble EGFP (pEGFP-N1) are also shown (bottom panels). DAPI, 4',6-diamidino-2-phenylindole; EGFP, Enhanced green fluorescent protein. Scale bar: 10 μm.

    Article Snippet: For the functional characterization of the COL4A3 signal-peptide deletion, the genomic DNA region coding for the predicted signal peptide was PCR amplified from the patient’s genomic DNA (with primers including Xho I and Kpn I restriction sites: COL4A3_SP_F 5’-ggcctcgaGGTGGCCTGAGAGCCTGA-3’ and COL4A3_SP_R 5’-agaggtaccTGGAGGAGGGATGGAAGTG-3’ ) and cloned in-frame upstream of the EGFP (Enhanced Green Fluorescent Protein) coding region in the pEGFP-N1 plasmid (Clontech, Mountain View, CA).

    Techniques: Functional Assay, Expressing, Positive Control

    Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. EGFP was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 ORF. The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.

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

    Article Title: UL40-mediated NK evasion during productive infection with human cytomegalovirus

    doi: 10.1073/pnas.112680099

    Figure Lengend Snippet: Generation of an HCMV strain AD169, UL40 deletion mutant, ΔUL40. ( A ) A schematic representation of the HCMV strain AD169 genome shows the inverted repeats and Hin dIII sites. EGFP was inserted into the viral genome under the control of the UL40 promoter while deleting most of the UL40 ORF. The relative positions of PCR primer sets 1 and 2 are shown. ( B ) PCR analysis of viral DNA showing the absence of a PCR product with the ΔUL40 template with primer set 1, consistent with a UL40 deletion. With primer set 2, the increased mobility of the DNA fragment generated by using the HCMVΔUL40 DNA, relative to the parental virus, corresponds to an appropriate EGFP insertion. Primer set C is a positive control amplifying a remote region of the genome within the β2.7 gene. ( C ) Growth curves showing no difference between parent strain, AD169, and ΔUL40. Human fetal foreskin fibroblasts were infected with either strain AD169 or ΔUL40 [multiplicity of infection (MOI) = 0.1] for 90 min at 37°C. At 3, 6, 9,12, and 15 days postinfection (pi), the tissue culture supernatant was harvested. The virus titer at each time point then was determined by a plaque assay on human fetal foreskin fibroblasts. The experiment was repeated with duplicate samples. pfu, plaque-forming units.

    Article Snippet: The UL40 promoter (nucleotides 54,329–53,879, terminating one nucleotide upstream of the translational start site) was amplified by PCR and fused to the EGFP ORF in pEGFP-N1 (CLONTECH).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Generated, Positive Control, Infection, Plaque Assay

    Expression of the intronic shINX1 plasmid selectively reduces Hve–inx1 mRNA levels and uncouples the AP neuron. A , B , Double labeling of embryonic AP neurons 48 h after expression of the EGFP–shINX1 reporter vector ( A1–A3 ; asterisks indicate AP neurons) and in situ hybridization (ISH; B1–B3 ), revealing the loss of INX1 mRNA staining (red arrowheads). C , D , Control expression of an shRNA targeting INX19 ( C1–C3 ; asterisks) fails to diminish INX1 mRNA labeling intensity ( D1–D3 ; red arrowheads). E , Quantification of INX1 mRNA staining intensity after shINX1 and INX19 expression. Values represent gray levels above background, corresponding to the location of the APs (transformed neurons; black bars) or nontransformed neurons adjacent to the APs (non-transformed neurons; gray bars). Data are ±SD (** p

    Journal: The Journal of Neuroscience

    Article Title: Gap Junction-Dependent Homolog Avoidance in the Developing CNS

    doi: 10.1523/JNEUROSCI.2387-13.2013

    Figure Lengend Snippet: Expression of the intronic shINX1 plasmid selectively reduces Hve–inx1 mRNA levels and uncouples the AP neuron. A , B , Double labeling of embryonic AP neurons 48 h after expression of the EGFP–shINX1 reporter vector ( A1–A3 ; asterisks indicate AP neurons) and in situ hybridization (ISH; B1–B3 ), revealing the loss of INX1 mRNA staining (red arrowheads). C , D , Control expression of an shRNA targeting INX19 ( C1–C3 ; asterisks) fails to diminish INX1 mRNA labeling intensity ( D1–D3 ; red arrowheads). E , Quantification of INX1 mRNA staining intensity after shINX1 and INX19 expression. Values represent gray levels above background, corresponding to the location of the APs (transformed neurons; black bars) or nontransformed neurons adjacent to the APs (non-transformed neurons; gray bars). Data are ±SD (** p

    Article Snippet: A short-hairpin RNA (shRNA) expression cassette was designed with a leech actin intron ( Hm Act1; GenBank accession number , base pairs 1801–1956) inserted downstream of the ORF of EGFP (pEGFP–C3; Clontech) between the BglII and BamHI sites of the multiple cloning region.

    Techniques: Expressing, Plasmid Preparation, Labeling, In Situ Hybridization, Staining, shRNA, Transformation Assay