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Southern blot of parental (HCT116) and knock-in clone (HCT116-Polε wt/exo- ) after Cre-mediated excision. Genomic DNA was digested with SacI and resolved on a 1% agarose gel in TBE. The DNA was transferred to <t>Hybond</t> N + membrane (Amersham) and blotted with probe against HA2 (shown in Figure 1—figure supplement 1 ). The sizes of the 1 kb ladder are shown to the left of the blot.
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1) Product Images from "Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair"

Article Title: Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

Journal: eLife

doi: 10.7554/eLife.32692

Southern blot of parental (HCT116) and knock-in clone (HCT116-Polε wt/exo- ) after Cre-mediated excision. Genomic DNA was digested with SacI and resolved on a 1% agarose gel in TBE. The DNA was transferred to Hybond N + membrane (Amersham) and blotted with probe against HA2 (shown in Figure 1—figure supplement 1 ). The sizes of the 1 kb ladder are shown to the left of the blot.
Figure Legend Snippet: Southern blot of parental (HCT116) and knock-in clone (HCT116-Polε wt/exo- ) after Cre-mediated excision. Genomic DNA was digested with SacI and resolved on a 1% agarose gel in TBE. The DNA was transferred to Hybond N + membrane (Amersham) and blotted with probe against HA2 (shown in Figure 1—figure supplement 1 ). The sizes of the 1 kb ladder are shown to the left of the blot.

Techniques Used: Southern Blot, Knock-In, Agarose Gel Electrophoresis

2) Product Images from "Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition"

Article Title: Role of Leishmania (Leishmania) chagasi amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-6-3

Expression of sense and antisense  Ldccys2  transcripts in  L. (L.) chagasi  and survival of wild type, sense and antisense  Ldccys2  expressing amastigotes in macrophages.  (A) Northern blot analyses of amastigotes expressing sense and antisense transcripts. Total RNA (10 μg/lane) isolated from axenic amastigotes of wildtype parasites, transfectants with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid were separated on 1.2% (w/v) formaldehyde agarose gel and blotted on to Hybond N+ membrane. Blots were probed with probes specific for antisense (panel 1) and sense (panel 2) transcripts. Panel 3 represents ethidium bromide stained gel. (B) Western blot analysis of sense and antisense Ldccys2 expressing amastigotes. Equal amounts of total protein extracted from axenically transformed amastigotes with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid and wildtype parasites were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond P membrane. The blot was probed with α-Ldccys2 antibody (panel 1) and a duplicate gel was stained with Coomassie blue (panel2). (C) Bar graph showing total number of amastigotes within the macrophage cells. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amstigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining.(D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.
Figure Legend Snippet: Expression of sense and antisense Ldccys2 transcripts in L. (L.) chagasi and survival of wild type, sense and antisense Ldccys2 expressing amastigotes in macrophages. (A) Northern blot analyses of amastigotes expressing sense and antisense transcripts. Total RNA (10 μg/lane) isolated from axenic amastigotes of wildtype parasites, transfectants with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid were separated on 1.2% (w/v) formaldehyde agarose gel and blotted on to Hybond N+ membrane. Blots were probed with probes specific for antisense (panel 1) and sense (panel 2) transcripts. Panel 3 represents ethidium bromide stained gel. (B) Western blot analysis of sense and antisense Ldccys2 expressing amastigotes. Equal amounts of total protein extracted from axenically transformed amastigotes with P6.5/Ldccys2 sense plasmid and P6.5/Ldccys2 antisense plasmid and wildtype parasites were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond P membrane. The blot was probed with α-Ldccys2 antibody (panel 1) and a duplicate gel was stained with Coomassie blue (panel2). (C) Bar graph showing total number of amastigotes within the macrophage cells. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amstigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining.(D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.

Techniques Used: Expressing, Northern Blot, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Western Blot, Transformation Assay, SDS Page, Infection, Diff-Quik

Ldccys2 is a single copy gene and is expressed only in amastigotes. (A) Southern blot hybridization of digested genomic DNA from L. (L.) chagasi (Lc) and L. (L.) donovani (Ld). 5 μg of genomic DNA was digested with restriction enzymes as mentioned in the figure and blotted onto Hybond-N membranes. The blot was probed with coding region pf Ldccys2 cDNA clone. (B) Northern blot analyses of L. (L.) chagasi total RNA. Total RNA (10 μg/lane) from promastigotes of logarithmic (lane 1), stationary (lane 2) growth phase, U937 cells (human macrophage cell line) infected with promastigotes for 96 h (lane 3) and uninfected U937 cells (lane 4) were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with PCR amplified DNA fragment containing 3'UTR from Ldccys2 (panel I), Ldccys1 (154 bp, near the polyA region, panel II), and coding region of α- tubulin from L. (L.) chagasi , a kind gift from Dr. M.E. Wilson (panel IV). (C) Western blot analysis of L. (L.) chagasi promastigotes and amastigotes. Equal amounts of proteins from promastigotes of logarithmic (lane 1), stationary (lane 2) phase, U937 cells infected with promastigotes (lane 3) and uninfected U937 cells (lane 4) were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond – P membrane. Western blot analysis was carried out using α-Ldccys2 antibody.
Figure Legend Snippet: Ldccys2 is a single copy gene and is expressed only in amastigotes. (A) Southern blot hybridization of digested genomic DNA from L. (L.) chagasi (Lc) and L. (L.) donovani (Ld). 5 μg of genomic DNA was digested with restriction enzymes as mentioned in the figure and blotted onto Hybond-N membranes. The blot was probed with coding region pf Ldccys2 cDNA clone. (B) Northern blot analyses of L. (L.) chagasi total RNA. Total RNA (10 μg/lane) from promastigotes of logarithmic (lane 1), stationary (lane 2) growth phase, U937 cells (human macrophage cell line) infected with promastigotes for 96 h (lane 3) and uninfected U937 cells (lane 4) were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with PCR amplified DNA fragment containing 3'UTR from Ldccys2 (panel I), Ldccys1 (154 bp, near the polyA region, panel II), and coding region of α- tubulin from L. (L.) chagasi , a kind gift from Dr. M.E. Wilson (panel IV). (C) Western blot analysis of L. (L.) chagasi promastigotes and amastigotes. Equal amounts of proteins from promastigotes of logarithmic (lane 1), stationary (lane 2) phase, U937 cells infected with promastigotes (lane 3) and uninfected U937 cells (lane 4) were separated on 10% (w/v) SDS-PAGE and blotted on to Hybond – P membrane. Western blot analysis was carried out using α-Ldccys2 antibody.

Techniques Used: Southern Blot, Hybridization, Northern Blot, Infection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Western Blot, SDS Page

Ldccys2  single allele gene replacement.  (A) Schematic representation of the wild type and  hyg/dhfr-ts  targeted alleles of  Ldccys2 . The location of the  Pst I sites used to characterize the hygromycin B resistant recombinants is shown by arrows. The bold lines labelled a, b and c represents the location of different probes used in Southern analyses. (B) Southern analyses of  Ldccys2  heterozygous knockout mutants. Genomic DNA (2 μg/lane) from wild type  L. (L.) chagasi  (WT) and  Ldccys2  heterozygous knockout mutants (KO) were digested with  Pst I and separated on a 0.9% (w/v) agarose gel. The DNA was blotted on to Hybond N+ membrane and hybridized with probes a (i), b (ii) and c (iii). Asterisks: 1- represents wild type allele of  Ldccys2  (3.5 kb), 2 and 3- represent disrupted allele (3.0 kb and 2.8 kb). The grey lines indicate the size of expected bands upon PstI digestion.
Figure Legend Snippet: Ldccys2 single allele gene replacement. (A) Schematic representation of the wild type and hyg/dhfr-ts targeted alleles of Ldccys2 . The location of the Pst I sites used to characterize the hygromycin B resistant recombinants is shown by arrows. The bold lines labelled a, b and c represents the location of different probes used in Southern analyses. (B) Southern analyses of Ldccys2 heterozygous knockout mutants. Genomic DNA (2 μg/lane) from wild type L. (L.) chagasi (WT) and Ldccys2 heterozygous knockout mutants (KO) were digested with Pst I and separated on a 0.9% (w/v) agarose gel. The DNA was blotted on to Hybond N+ membrane and hybridized with probes a (i), b (ii) and c (iii). Asterisks: 1- represents wild type allele of Ldccys2 (3.5 kb), 2 and 3- represent disrupted allele (3.0 kb and 2.8 kb). The grey lines indicate the size of expected bands upon PstI digestion.

Techniques Used: Knock-Out, Agarose Gel Electrophoresis

Characterization of  L. (L.) chagasi  heterozygous knockout mutant amastigotes and intra macrophage survival of  Ldccys2KO  amastigotes  in vitro .  (A) Northern blot analysis. Total RNA (10 μg/lane) from U937 cells infected with wild type promastigotes,  Ldccys2KO  and uninfected U937 cells were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with  Ldccys2  coding region DNA probe (1) and α-tubulin gene from  L. (L.) chagasi  (2). (B) Western blot analysis. Equal amount of lysates from U937 cells infected with wild type promastigotes,  Ldccys2KO  promastigotes and uninfected U937 cells were separated on 10% (w/v) SDS-PAGE, blotted on to Hybond P membrane. Panel 1 represents the membrane that was probed with α-Ldccys2 antibody and panel 2 is the duplicate gel stained with Coomassie blue. (C) Bar graph showing the number of intracellular amastigotes. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amastigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining. (D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.
Figure Legend Snippet: Characterization of L. (L.) chagasi heterozygous knockout mutant amastigotes and intra macrophage survival of Ldccys2KO amastigotes in vitro . (A) Northern blot analysis. Total RNA (10 μg/lane) from U937 cells infected with wild type promastigotes, Ldccys2KO and uninfected U937 cells were separated on 1.2% (w/v) formaldehyde agarose gel and transferred on to Hybond N+ membrane. Blot was hybridized with Ldccys2 coding region DNA probe (1) and α-tubulin gene from L. (L.) chagasi (2). (B) Western blot analysis. Equal amount of lysates from U937 cells infected with wild type promastigotes, Ldccys2KO promastigotes and uninfected U937 cells were separated on 10% (w/v) SDS-PAGE, blotted on to Hybond P membrane. Panel 1 represents the membrane that was probed with α-Ldccys2 antibody and panel 2 is the duplicate gel stained with Coomassie blue. (C) Bar graph showing the number of intracellular amastigotes. U937 macrophage cells were infected at a macrophage to parasite ratio of 1:10. The survival of the amastigotes within the macrophages was evaluated every 12 hours by cytospin and Diff-Quick staining. (D) Graph showing the percent of total macrophages infected at the given time points. For each treatment, 100 infected macrophages were counted. Values represent means ± SEM from three independent experiments.

Techniques Used: Knock-Out, Mutagenesis, In Vitro, Northern Blot, Infection, Agarose Gel Electrophoresis, Western Blot, SDS Page, Staining, Diff-Quik

3) Product Images from "Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development"

Article Title: Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development

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

doi:

DNA blot analysis of the genomic DNA methylation in the CASH plants and an antisense MET1 plant. Total genomic DNA (2 μg) was digested with Hpa II and transferred onto Hybond-N+ membrane. ( A ). ( B ).
Figure Legend Snippet: DNA blot analysis of the genomic DNA methylation in the CASH plants and an antisense MET1 plant. Total genomic DNA (2 μg) was digested with Hpa II and transferred onto Hybond-N+ membrane. ( A ). ( B ).

Techniques Used: DNA Methylation Assay

4) Product Images from "Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants"

Article Title: Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants

Journal: Turkish Journal of Biology

doi: 10.3906/biy-1705-30

Molecular verification of transgenes in tobacco plants. a) Schematic representation of the T-DNA region of pCam: MsDJLP, the expression vector used for genetic transformation. CaMV 35S: the cauliflower mosaic 35S promoter; MsDJLP: Medicago sativa DnaJ-like protein; HPT: hygromycin phosphotransferase. b) Gel image of the PCR amplification of 0.8-kb HPT gene. c) PCR band of 0.495-kb MsDJLP fragments in tobacco transformants. Mw: molecular weight; P: plasmid DNA of expression vector pCam: MsDJLP; WT: wild-type plant. d) Southern blot analysis of tobacco plants. Ten micrograms of genomic DNA was digested with Hind III, the size-fractionated DNA fragments were transferred to a Hybond N + membrane, and blots were hybridized with α 32 P-labeled PCR probe amplified from MsDJLP.
Figure Legend Snippet: Molecular verification of transgenes in tobacco plants. a) Schematic representation of the T-DNA region of pCam: MsDJLP, the expression vector used for genetic transformation. CaMV 35S: the cauliflower mosaic 35S promoter; MsDJLP: Medicago sativa DnaJ-like protein; HPT: hygromycin phosphotransferase. b) Gel image of the PCR amplification of 0.8-kb HPT gene. c) PCR band of 0.495-kb MsDJLP fragments in tobacco transformants. Mw: molecular weight; P: plasmid DNA of expression vector pCam: MsDJLP; WT: wild-type plant. d) Southern blot analysis of tobacco plants. Ten micrograms of genomic DNA was digested with Hind III, the size-fractionated DNA fragments were transferred to a Hybond N + membrane, and blots were hybridized with α 32 P-labeled PCR probe amplified from MsDJLP.

Techniques Used: Expressing, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Amplification, Molecular Weight, Southern Blot, Labeling

5) Product Images from "GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION"

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION

Journal: The Journal of surgical research

doi: 10.1016/j.jss.2009.03.072

Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.
Figure Legend Snippet: Time course of IL-8 mRNA induction by EGF and /or BBS. A) Autoradiography of Northern blots demonstrates steady state levels of IL-8 mRNA after treatment with EGF (1 ng/ml), BBS (100nM), or both EGF and BBS. Total RNA (15 μg/lane) was isolated from MDA-MB-231 cells at the indicated time points after treatment. RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto a Hybond-N+ membrane, and hybridized with a [α 32 P]dATP-labeled cDNA probe. Equal loading and transfer was verified by rehybridizing the blot with a radiolabeled probe for 18S RNAse. B) Quantitative real-time PCR analysis of IL-8 mRNA levels showing synergistic increase at 4 h. As a negative control, mRNA from SK-BR-3 cells was used.

Techniques Used: Autoradiography, Northern Blot, Isolation, Multiple Displacement Amplification, Labeling, Real-time Polymerase Chain Reaction, Negative Control

6) Product Images from "Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide"

Article Title: Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0702757

Northern blot analysis of SGHPL-4 cells. RNA was isolated from SGHPL-4 cells following a 6 h incubation in the absence (lane 1) or presence of IL-1β and TNFα (each at 5 ng ml −1 ; lane 2) or HGF (10 ng ml −1 ; lane 3; 100 ng ml −1 ; lane 4). After separation by electrophoresis through a 1.5% agarose gel the RNA was transferred to Hybond N+ membrane and a 3.6  kb band (shown by arrow) detected following hybridization with a  32 P-labelled probe for human iNOS.
Figure Legend Snippet: Northern blot analysis of SGHPL-4 cells. RNA was isolated from SGHPL-4 cells following a 6 h incubation in the absence (lane 1) or presence of IL-1β and TNFα (each at 5 ng ml −1 ; lane 2) or HGF (10 ng ml −1 ; lane 3; 100 ng ml −1 ; lane 4). After separation by electrophoresis through a 1.5% agarose gel the RNA was transferred to Hybond N+ membrane and a 3.6  kb band (shown by arrow) detected following hybridization with a 32 P-labelled probe for human iNOS.

Techniques Used: Northern Blot, Isolation, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Hybridization

7) Product Images from "Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556"

Article Title: Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042769

Southern blot and PCR analyses of M. smegmatis LS2 strains. The genomic DNA was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to Hybond-N + membrane and hybridized by MSMEG_1556 probe. A. The expected DNA fragments hybridized by MSMEG_1556 probe were 2.12 kb and 2.55 kb. The expected size of PCR product from M. smegmatis LS2 strain was 3.15 kb. B. The expected DNA fragments hybridized by MSMEG_1556 probe was 3.37 kb and the expected size of PCR product from M. smegmatis mc 2 155 strain was 1.88 kb. C. Confirmation of M. smegmatis LS2 strains by Southern blot. Lanes 1–5. M. smegmatis LS2 strains have the hybridized DNA bands of 2.12 kb and 2.55 kb; lane 6. wild type M. smegmatis mc 2 155 has the hybridized DNA band of 3.37 kb. D. Confirmation of M. smegmatis LS2 strains by PCR. Lanes 1–5. the PCR product (3.15 kb) from M. smegmatis LS2 strains; lane 6. the PCR product (1.88 kb) from wild type M. smegmatis mc 2 155.
Figure Legend Snippet: Southern blot and PCR analyses of M. smegmatis LS2 strains. The genomic DNA was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to Hybond-N + membrane and hybridized by MSMEG_1556 probe. A. The expected DNA fragments hybridized by MSMEG_1556 probe were 2.12 kb and 2.55 kb. The expected size of PCR product from M. smegmatis LS2 strain was 3.15 kb. B. The expected DNA fragments hybridized by MSMEG_1556 probe was 3.37 kb and the expected size of PCR product from M. smegmatis mc 2 155 strain was 1.88 kb. C. Confirmation of M. smegmatis LS2 strains by Southern blot. Lanes 1–5. M. smegmatis LS2 strains have the hybridized DNA bands of 2.12 kb and 2.55 kb; lane 6. wild type M. smegmatis mc 2 155 has the hybridized DNA band of 3.37 kb. D. Confirmation of M. smegmatis LS2 strains by PCR. Lanes 1–5. the PCR product (3.15 kb) from M. smegmatis LS2 strains; lane 6. the PCR product (1.88 kb) from wild type M. smegmatis mc 2 155.

Techniques Used: Southern Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis

8) Product Images from "Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines"

Article Title: Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601683

An example of a Northern blot for OPN and S100A4 mRNA levels in the clonal cell lines. The following samples were electrophoresed through 0.8% (w v −1 ) formaldehyde agarose gels and blotted onto Hybond-N membrane: Rama 800 (lane 1), R37C9Pooled (lane 2), R37C9VM13 (lane 3), R37C9VM16 (lane 4), R37C9VM8 (lane 5), R37C9VM6 (lane 6), R37C9VM25 (lane 7), Rama 37 (lane 8), R37C9VM29 (lane 9), R37C9VM14 (lane 10), R37C9VM3 (lane 11), R37C9VM18 (lane 12). In panel ( A ) the membrane was incubated with α 32 P-labelled OPN cDNA and subjected to autoradiography. In panel ( B ) the membrane was incubated with α 32 P-labelled S100A4 cDNA and subjected to autoradiography. In panel ( C ) the ethidium bromide-stained 18S and 28S ribosomal RNA bands on the membrane before hybridisation were photographed. The 1.6 kb OPN mRNA, 0.8 kb S100A4 mRNA and 18S and 28S ribosomal RNAs are shown. ( D ) Graph of OPN mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative OPN mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( E ) S100A4 mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative S100A4 mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( D , E ) Contain results for 9 C9-Met-DNA-transfected Rama 37 cell lines plus the uncloned C9-Met-DNA-transfected cells and the parental Rama 37 cells.
Figure Legend Snippet: An example of a Northern blot for OPN and S100A4 mRNA levels in the clonal cell lines. The following samples were electrophoresed through 0.8% (w v −1 ) formaldehyde agarose gels and blotted onto Hybond-N membrane: Rama 800 (lane 1), R37C9Pooled (lane 2), R37C9VM13 (lane 3), R37C9VM16 (lane 4), R37C9VM8 (lane 5), R37C9VM6 (lane 6), R37C9VM25 (lane 7), Rama 37 (lane 8), R37C9VM29 (lane 9), R37C9VM14 (lane 10), R37C9VM3 (lane 11), R37C9VM18 (lane 12). In panel ( A ) the membrane was incubated with α 32 P-labelled OPN cDNA and subjected to autoradiography. In panel ( B ) the membrane was incubated with α 32 P-labelled S100A4 cDNA and subjected to autoradiography. In panel ( C ) the ethidium bromide-stained 18S and 28S ribosomal RNA bands on the membrane before hybridisation were photographed. The 1.6 kb OPN mRNA, 0.8 kb S100A4 mRNA and 18S and 28S ribosomal RNAs are shown. ( D ) Graph of OPN mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative OPN mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( E ) S100A4 mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative S100A4 mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( D , E ) Contain results for 9 C9-Met-DNA-transfected Rama 37 cell lines plus the uncloned C9-Met-DNA-transfected cells and the parental Rama 37 cells.

Techniques Used: Northern Blot, Incubation, Autoradiography, Staining, Hybridization, Clone Assay, In Vivo, Transfection

An example of Southern hybridisation of C9-Met-DNA to DNA obtained from the clonal cell lines. ( A ) DNA from each of the cell lines was digested with Eco R1, electrophoresed on agarose gels, and blotted onto Hybond-N membrane. The samples loaded on the gel were 500 copies C9-Met-DNA (lane 1), 50 copies C9-Met-DNA (lane 2), Rama 37 DNA (lane 3), R37C9VM18 (lane 4), R37C9VM13 (lane 5), R37C9VM6 (lane 6), R37C9VM29 (lane 7), R37C9VM14 (lane 8), R37C9VM3 (lane 9), R37C9VM8 (lane 10), R37C9VM16 (lane 11) and R37C9VM25 (lane 12). The membrane was incubated with α 32 P-labelled C9-Met-DNA under hybridising conditions and subjected to autoradiography. The position of the authentic C9-Met-DNA is shown by the arrow. ( B ) Graph of C9-Met-DNA copy number (copy no) of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). Results for the nine cloned C9-Met-DNA-transfected cell lines plus Rama 37 parental cells are shown.
Figure Legend Snippet: An example of Southern hybridisation of C9-Met-DNA to DNA obtained from the clonal cell lines. ( A ) DNA from each of the cell lines was digested with Eco R1, electrophoresed on agarose gels, and blotted onto Hybond-N membrane. The samples loaded on the gel were 500 copies C9-Met-DNA (lane 1), 50 copies C9-Met-DNA (lane 2), Rama 37 DNA (lane 3), R37C9VM18 (lane 4), R37C9VM13 (lane 5), R37C9VM6 (lane 6), R37C9VM29 (lane 7), R37C9VM14 (lane 8), R37C9VM3 (lane 9), R37C9VM8 (lane 10), R37C9VM16 (lane 11) and R37C9VM25 (lane 12). The membrane was incubated with α 32 P-labelled C9-Met-DNA under hybridising conditions and subjected to autoradiography. The position of the authentic C9-Met-DNA is shown by the arrow. ( B ) Graph of C9-Met-DNA copy number (copy no) of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). Results for the nine cloned C9-Met-DNA-transfected cell lines plus Rama 37 parental cells are shown.

Techniques Used: Hybridization, Incubation, Autoradiography, In Vivo, Clone Assay, Transfection

9) Product Images from "MOS11: A New Component in the mRNA Export Pathway"

Article Title: MOS11: A New Component in the mRNA Export Pathway

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1001250

mRNA export is impaired in mos11 plants. (A) Morphology of 4-week-old soil-grown WT, mos11-1 and mos11-2 plants. (B) Dot blot of WT, mos11-1 and mos11-2 . The same amount of WT, mos11-1 , and mos11-2 total RNA were applied to the Hybond-N+ membrane and hybridized by 32 P-ATP labeled 18-mer oligo dT. The amount of total RNA loaded to each dot was indicated on the right of the blot. The radioactive signal was detected by a phosphor-imager. (C) Whole mount in situ mRNA localization of 7-day-old seedlings probed with 48-mer oligo d(T) labeled with Alexa 488. Observations were done at 63× magnification and microscope settings were identical for all samples.
Figure Legend Snippet: mRNA export is impaired in mos11 plants. (A) Morphology of 4-week-old soil-grown WT, mos11-1 and mos11-2 plants. (B) Dot blot of WT, mos11-1 and mos11-2 . The same amount of WT, mos11-1 , and mos11-2 total RNA were applied to the Hybond-N+ membrane and hybridized by 32 P-ATP labeled 18-mer oligo dT. The amount of total RNA loaded to each dot was indicated on the right of the blot. The radioactive signal was detected by a phosphor-imager. (C) Whole mount in situ mRNA localization of 7-day-old seedlings probed with 48-mer oligo d(T) labeled with Alexa 488. Observations were done at 63× magnification and microscope settings were identical for all samples.

Techniques Used: Dot Blot, Labeling, In Situ, Microscopy

10) Product Images from "Cambial-Region-Specific Expression of the Agrobacterium iaa Genes in Transgenic Aspen Visualized by a Linked uidA Reporter Gene 1"

Article Title: Cambial-Region-Specific Expression of the Agrobacterium iaa Genes in Transgenic Aspen Visualized by a Linked uidA Reporter Gene 1

Journal: Plant Physiology

doi:

Southern-blot analysis of hybrid aspen lines regenerated after transformation with the vector p812C1C. Ten micrograms of genomic DNA, digested with various restriction enzymes, was loaded on each lane of a agarose gel, transferred to a Hybond-N membrane, and probed with 32 P-labeled DNA from either the iaaM or the uidA gene. a, Digestion with Xba I and hybridization with an iaaM -specific probe. b, Digestion with Sac I and hybridization with an uidA -specific probe. c, Digestion with Eco RV and hybridization with an uidA -specific probe. Letters on the top of the figure (A–O) indicate the different lines. The copy number of the inserted T-DNA(s) is shown in parentheses.
Figure Legend Snippet: Southern-blot analysis of hybrid aspen lines regenerated after transformation with the vector p812C1C. Ten micrograms of genomic DNA, digested with various restriction enzymes, was loaded on each lane of a agarose gel, transferred to a Hybond-N membrane, and probed with 32 P-labeled DNA from either the iaaM or the uidA gene. a, Digestion with Xba I and hybridization with an iaaM -specific probe. b, Digestion with Sac I and hybridization with an uidA -specific probe. c, Digestion with Eco RV and hybridization with an uidA -specific probe. Letters on the top of the figure (A–O) indicate the different lines. The copy number of the inserted T-DNA(s) is shown in parentheses.

Techniques Used: Southern Blot, Transformation Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Labeling, Hybridization

11) Product Images from "Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato"

Article Title: Highly efficient homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

Journal: bioRxiv

doi: 10.1101/521419

Southern Blot analysis of the ANT1 edited locus. Genomic DNAs were isolated from the leaves of the ANT1 HDR events and 20 µg of each gDNAs were digested with NsiI and resolved on 0.8% agrose gel. The resolved DNAs were then blotted onto Hybond N+ membrane and later detected using Dioxigenin labeled Probe (  Supplemental Figure 2 ). The marker lane (M, DNA Molecular Weight Marker III, Digoxigenin-labeled, Roche) is separated for better illustration of the image. The expected bands with sizes are denoted beside the Southern blot panel. C1.4.4, C1.9.1, C1.10.2, C1.12.3: GE1 lines showed strong amplification of RJ and LJ in   Supplemental Figure 9 ; C9.1.1 (RJ and LJ edited) and C9.3.1 (only RJ inserted): two GE1 line obtained by pTC217 tool.
Figure Legend Snippet: Southern Blot analysis of the ANT1 edited locus. Genomic DNAs were isolated from the leaves of the ANT1 HDR events and 20 µg of each gDNAs were digested with NsiI and resolved on 0.8% agrose gel. The resolved DNAs were then blotted onto Hybond N+ membrane and later detected using Dioxigenin labeled Probe ( Supplemental Figure 2 ). The marker lane (M, DNA Molecular Weight Marker III, Digoxigenin-labeled, Roche) is separated for better illustration of the image. The expected bands with sizes are denoted beside the Southern blot panel. C1.4.4, C1.9.1, C1.10.2, C1.12.3: GE1 lines showed strong amplification of RJ and LJ in Supplemental Figure 9 ; C9.1.1 (RJ and LJ edited) and C9.3.1 (only RJ inserted): two GE1 line obtained by pTC217 tool.

Techniques Used: Southern Blot, Isolation, Labeling, Marker, Molecular Weight, Amplification

12) Product Images from "Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from Borrelia turicatae, a Causative Agent of Relapsing Fever"

Article Title: Cloning and Molecular Characterization of a Multicopy, Linear Plasmid-Carried, Repeat Motif-Containing Gene from Borrelia turicatae, a Causative Agent of Relapsing Fever

Journal: Journal of Bacteriology

doi:

Identification of the genomic elements carrying repA by 2D PFGE and Southern hybridization. DNA liberated from B. turicatae and B. parkeri was fractionated by 2D CHEF-PFGE as described in the text. The DNA was visualized by ethidium bromide staining and was photographed (left panel). The DNA was then transferred onto Hybond N membranes and hybridized with a PCR-generated probe as described in the text. The probe targets almost the entire repA gene and was generated with the repAF1 and repAR1 primers and pBT2.2, the repA -carrying recombinant plasmid, as amplification template. Molecular size standards (lambda monocuts) are indicated between the panels. The isolates analyzed and the direction of electrophoresis in each dimension are indicated.
Figure Legend Snippet: Identification of the genomic elements carrying repA by 2D PFGE and Southern hybridization. DNA liberated from B. turicatae and B. parkeri was fractionated by 2D CHEF-PFGE as described in the text. The DNA was visualized by ethidium bromide staining and was photographed (left panel). The DNA was then transferred onto Hybond N membranes and hybridized with a PCR-generated probe as described in the text. The probe targets almost the entire repA gene and was generated with the repAF1 and repAR1 primers and pBT2.2, the repA -carrying recombinant plasmid, as amplification template. Molecular size standards (lambda monocuts) are indicated between the panels. The isolates analyzed and the direction of electrophoresis in each dimension are indicated.

Techniques Used: Hybridization, Staining, Polymerase Chain Reaction, Generated, Recombinant, Plasmid Preparation, Amplification, Electrophoresis

13) Product Images from "Acquired Thermotolerance and Expression of the HSP100/ClpB Genes of Lima Bean 1"

Article Title: Acquired Thermotolerance and Expression of the HSP100/ClpB Genes of Lima Bean 1

Journal: Plant Physiology

doi:

Northern blot of total RNA. A, RNA was extracted from leaf tissues of 3- to 4-week-old lima bean JWAR and F1072. Three- to 4-week-old seedlings were incubated at 40°C for 0, 30, 60, 120, or 300 min. RNA was extracted from immature leaves, separated by formaldehyde, agarose gel electrophoresis, and blotted onto Hybond N + (Amersham, Arlington Heights, IL) membrane. The northern blot was hybridized with antisense riboprobe derived from lima bean cyt HSP100/ClpB gene fragment and an actin gene fragment simultaneously. B, Quantification of hybridizing signals. An autoradiograph of the hybridized northern blot in A was converted to a digital image and the density of the bands measured using the NIH Image program. Relative densities were adjusted based on the density of the actin signal at that time point. The least actin signal was set at a value of 1.0, and all of the other actin signals adjusted to this signal. The actin ratio was then used to normalize the relative density of the signal from the HSP100 probe for the same sample.
Figure Legend Snippet: Northern blot of total RNA. A, RNA was extracted from leaf tissues of 3- to 4-week-old lima bean JWAR and F1072. Three- to 4-week-old seedlings were incubated at 40°C for 0, 30, 60, 120, or 300 min. RNA was extracted from immature leaves, separated by formaldehyde, agarose gel electrophoresis, and blotted onto Hybond N + (Amersham, Arlington Heights, IL) membrane. The northern blot was hybridized with antisense riboprobe derived from lima bean cyt HSP100/ClpB gene fragment and an actin gene fragment simultaneously. B, Quantification of hybridizing signals. An autoradiograph of the hybridized northern blot in A was converted to a digital image and the density of the bands measured using the NIH Image program. Relative densities were adjusted based on the density of the actin signal at that time point. The least actin signal was set at a value of 1.0, and all of the other actin signals adjusted to this signal. The actin ratio was then used to normalize the relative density of the signal from the HSP100 probe for the same sample.

Techniques Used: Northern Blot, Incubation, Agarose Gel Electrophoresis, Derivative Assay, Autoradiography

14) Product Images from "Cap-Independent Translational Enhancement by the 3? Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1"

Article Title: Cap-Independent Translational Enhancement by the 3? Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1

Journal: Journal of Virology

doi: 10.1128/JVI.77.22.12113-12121.2003

Immunodetection of capped RNA with a cap-specific antibody. (A) Virion RNAs of TMGMV, BBMV, and RCNMV were fractionated on a denatured agarose gel, transferred onto a Hybond N+ membrane, and reacted with a cap-specific antibody. The concentration of each virion RNA was adjusted to load the same molar amount (1.5 pmol) of TMGMV genomic RNA, BBMV RNA3, and RCNMV RNA1. Identities of the RNAs are indicated on the left. (B) Total virion RNAs blotted onto the membrane used for immunoassay were detected with methylene blue staining.
Figure Legend Snippet: Immunodetection of capped RNA with a cap-specific antibody. (A) Virion RNAs of TMGMV, BBMV, and RCNMV were fractionated on a denatured agarose gel, transferred onto a Hybond N+ membrane, and reacted with a cap-specific antibody. The concentration of each virion RNA was adjusted to load the same molar amount (1.5 pmol) of TMGMV genomic RNA, BBMV RNA3, and RCNMV RNA1. Identities of the RNAs are indicated on the left. (B) Total virion RNAs blotted onto the membrane used for immunoassay were detected with methylene blue staining.

Techniques Used: Immunodetection, Agarose Gel Electrophoresis, Concentration Assay, Staining

15) Product Images from "Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast"

Article Title: Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt273

Effects of overproducing Din7 and Mhr1 on the levels of molecular species of HS ρ − mtDNA. Wild-type HS ρ − cells harboring the control plasmid pYES2/CT, the MHR1 expression plasmid pYESMHR1, the DIN7 expression plasmid pYES2/CT-DIN7 or the pESC-DIN7+MHR1 plasmid expressing both DIN7 and MHR1 were cultivated for 11 h at 30°C in RGal medium to induce gene expression. Whole cellular DNA was isolated from the cells, and ∼20 µg of cellular DNA was separated by 2D gel electrophoresis and transferred to a Hybond-N + membrane. HS ρ − mtDNA on the membrane was detected by Southern hybridization analysis using 32 P-labeled HS ρ − mtDNA as a probe. ( A -a) Schematic diagram of the molecular species of HS ρ − mtDNA separated by 2D gel electrophoresis. The first dimension separates DNA species according to mass, whereas the second dimension, which is run in the presence of ethidium bromide, fractionates molecules according to both mass and shape. The signals of the DNA species are indicated: continuous arc, concatemers (linear tandem multimers of varying continuous lengths); ssDNA, single-stranded DNA. Signals for supercoiled circular and nicked-circular double-stranded monomers and multimers were assigned by comparing A-e and A-f as described in the ‘Results’ section. 1C, 2C, 3C, 4C and 5C represent supercoiled closed-circular monomers, dimers, trimers, tetramers and pentamers, respectively. 1N, 2N, 3N, 4N and 5N represent nicked-circular monomers, dimers, trimers, tetramers and pentamers, respectively. Circular multimers are products of crossing-over–type homologous recombination ( 5 , 36 ). Signals corresponding to concatemers, supercoiled and nicked-circular monomers were measured and corrected by subtracting the background of an area of equivalent size that lacked radioactivity. (A-b, A-c, A-d and A-e) 2D gel profiles of HS ρ − mtDNA that were isolated from wild-type cells harbor ing pYES2/CT, pYESMHR1, pYES2/CT-DIN7 and pESC-DIN7+MHR1, respectively. ( B ) The top shows the amounts of concatemers relative to supercoiled closed circular monomers. The middle shows the amounts of single-stranded DNA molecules relative to supercoiled closed-circular monomers. The bottom shows the amounts of circular multimers relative to concatemers. The numbers obtained from these calculation were directly plotted. ( C ) The bottom is an example of a 1D gel profile of BglII-digested DNA with probes for 32 P-labeled HS ρ − mtDNA and the NUC1 gene, as described for Figure 2 B. The DSBs at ori5 (top) and the HS ρ − mtDNA copy number (middle) were calculated, normalized against those obtained from wild-type cells harboring HS ρ − mtDNA and plotted as described in Figure 2 C. (B and C) Each bar represents the average of three independent experiments. ( D ) Levels of overproduced Din7 and Mhr1 as measured by immunoblot analysis. Mitochondrial extracts were prepared from wild-type HS ρ − cells harboring the control plasmid pYES2/CT, the MHR1 expression plasmid pYESMHR1, the DIN7 expression plasmid pYES2/CT-DIN7 or the pESC-DIN7 + MHR1 plasmid, which were cultivated for 11 h at 30°C in 2 l of RGal medium to induce gene expression. Din7 and Mhr1 expression levels were detected by immunoblot analysis using anti-Din7 rabbit serum and anti-Mhr1 rabbit serum, respectively. As a control, porin was detected using a monoclonal anti-porin antibody.
Figure Legend Snippet: Effects of overproducing Din7 and Mhr1 on the levels of molecular species of HS ρ − mtDNA. Wild-type HS ρ − cells harboring the control plasmid pYES2/CT, the MHR1 expression plasmid pYESMHR1, the DIN7 expression plasmid pYES2/CT-DIN7 or the pESC-DIN7+MHR1 plasmid expressing both DIN7 and MHR1 were cultivated for 11 h at 30°C in RGal medium to induce gene expression. Whole cellular DNA was isolated from the cells, and ∼20 µg of cellular DNA was separated by 2D gel electrophoresis and transferred to a Hybond-N + membrane. HS ρ − mtDNA on the membrane was detected by Southern hybridization analysis using 32 P-labeled HS ρ − mtDNA as a probe. ( A -a) Schematic diagram of the molecular species of HS ρ − mtDNA separated by 2D gel electrophoresis. The first dimension separates DNA species according to mass, whereas the second dimension, which is run in the presence of ethidium bromide, fractionates molecules according to both mass and shape. The signals of the DNA species are indicated: continuous arc, concatemers (linear tandem multimers of varying continuous lengths); ssDNA, single-stranded DNA. Signals for supercoiled circular and nicked-circular double-stranded monomers and multimers were assigned by comparing A-e and A-f as described in the ‘Results’ section. 1C, 2C, 3C, 4C and 5C represent supercoiled closed-circular monomers, dimers, trimers, tetramers and pentamers, respectively. 1N, 2N, 3N, 4N and 5N represent nicked-circular monomers, dimers, trimers, tetramers and pentamers, respectively. Circular multimers are products of crossing-over–type homologous recombination ( 5 , 36 ). Signals corresponding to concatemers, supercoiled and nicked-circular monomers were measured and corrected by subtracting the background of an area of equivalent size that lacked radioactivity. (A-b, A-c, A-d and A-e) 2D gel profiles of HS ρ − mtDNA that were isolated from wild-type cells harbor ing pYES2/CT, pYESMHR1, pYES2/CT-DIN7 and pESC-DIN7+MHR1, respectively. ( B ) The top shows the amounts of concatemers relative to supercoiled closed circular monomers. The middle shows the amounts of single-stranded DNA molecules relative to supercoiled closed-circular monomers. The bottom shows the amounts of circular multimers relative to concatemers. The numbers obtained from these calculation were directly plotted. ( C ) The bottom is an example of a 1D gel profile of BglII-digested DNA with probes for 32 P-labeled HS ρ − mtDNA and the NUC1 gene, as described for Figure 2 B. The DSBs at ori5 (top) and the HS ρ − mtDNA copy number (middle) were calculated, normalized against those obtained from wild-type cells harboring HS ρ − mtDNA and plotted as described in Figure 2 C. (B and C) Each bar represents the average of three independent experiments. ( D ) Levels of overproduced Din7 and Mhr1 as measured by immunoblot analysis. Mitochondrial extracts were prepared from wild-type HS ρ − cells harboring the control plasmid pYES2/CT, the MHR1 expression plasmid pYESMHR1, the DIN7 expression plasmid pYES2/CT-DIN7 or the pESC-DIN7 + MHR1 plasmid, which were cultivated for 11 h at 30°C in 2 l of RGal medium to induce gene expression. Din7 and Mhr1 expression levels were detected by immunoblot analysis using anti-Din7 rabbit serum and anti-Mhr1 rabbit serum, respectively. As a control, porin was detected using a monoclonal anti-porin antibody.

Techniques Used: Plasmid Preparation, Expressing, Isolation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Hybridization, Labeling, Homologous Recombination, Radioactivity

Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. ( A ) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ − mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. ( B ) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ − mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA ( ∼ 10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N + membranes (GE Healthcare). The HS ρ − mtDNA on the membranes was detected by Southern hybridization using 32 P-labeled HS ρ − mtDNA and the NUC1 gene as probes, as described previously ( 5 ). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ − mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. ( C ) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ − mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ − mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ − mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ − mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. ( D ) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ – mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.
Figure Legend Snippet: Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. ( A ) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ − mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. ( B ) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ − mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA ( ∼ 10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N + membranes (GE Healthcare). The HS ρ − mtDNA on the membranes was detected by Southern hybridization using 32 P-labeled HS ρ − mtDNA and the NUC1 gene as probes, as described previously ( 5 ). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ − mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. ( C ) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ − mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ − mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ − mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ − mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. ( D ) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ – mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.

Techniques Used: Mutagenesis, Sequencing, Isolation, Electrophoresis, Agarose Gel Electrophoresis, Hybridization, Labeling, Derivative Assay, Generated, Expressing

16) Product Images from "Expression of ?-Expansin and Expansin-Like Genes in Deepwater Rice 1"

Article Title: Expression of ?-Expansin and Expansin-Like Genes in Deepwater Rice 1

Journal: Plant Physiology

doi: 10.1104/pp.008888

DNA gel-blot analyses showing the specificity of the gene-specific probes. Genomic DNA was digested with Eco RI (E), Xba I (X), Hin dIII (H), Sac I (S), Eco RI and Xba I (E+X), or with Hin dIII and Sac I (H+S). The digested DNA was separated by gel electrophoresis, blotted onto a Hybond N + membrane, and hybridized to the gene-specific probes indicated under each blot under the same conditions as described for RNA gel-blot analysis. We are only showing the DNA gel-blot analysis of those genes whose expression was detected in deepwater rice.
Figure Legend Snippet: DNA gel-blot analyses showing the specificity of the gene-specific probes. Genomic DNA was digested with Eco RI (E), Xba I (X), Hin dIII (H), Sac I (S), Eco RI and Xba I (E+X), or with Hin dIII and Sac I (H+S). The digested DNA was separated by gel electrophoresis, blotted onto a Hybond N + membrane, and hybridized to the gene-specific probes indicated under each blot under the same conditions as described for RNA gel-blot analysis. We are only showing the DNA gel-blot analysis of those genes whose expression was detected in deepwater rice.

Techniques Used: Western Blot, Nucleic Acid Electrophoresis, Expressing

17) Product Images from "Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1"

Article Title: Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

Journal: Current Genetics

doi: 10.1007/s00294-010-0313-3

Gene expression in the tra1-L3733A strain. a The promoter region of PHO5 was cloned as a LacZ reporter fusion into the LEU2 centromeric plasmid YCp87 and transformed into yeast strains CY4353 ( TRA1 ), CY4318 ( tra1 - A3727S ), CY4103 ( tra1 - L3733A ), CY4324 ( tra1 - F3740A ) and CY4350 ( tra1 - F3744A ), containing a plasmid copy of YHR100C. β-Galactosidase activity was determined after growth in low phosphate media for 15 h at 30°. Expression is shown as a percentage of that found for CY4353. Measurements were made in triplicate with the standard deviation indicated. b Expression from stress response elements. A cassette of two STRE elements was cloned into the Eco RI and Sac I sites of his3 - Δ88 - LacZ (Brandl et al. 1993 ) to give STRE - his3 - LacZ . These elements replace the normal Gcn4 binding site in HIS3 . STRE - his3 - LacZ and his3 - Δ88 were transformed into CY2706 and CY3003, and β-galactosidase assays performed after growth of cells in YPD containing 4% ethanol. c Expression levels determined by gene profiling (GP, mRNA-Seq) as compared with RNA dot blots ( ADE17 and RPL4a/b ) and LacZ reporter fusions ( PHO5 and RPL35a ). RNA was prepared from yeast strains CY3003 ( tra1 - L3733A ) and CY2706 ( TRA1 WT ). mRNA-Seq libraries were constructed and sequencing performed on the Illumina/Solexa Genome Analyzer II platform. Comparisons between the strains were made as outlined in “ Materials and methods ”. Similarly prepared RNA was spotted onto Hybond-N membrane and probed with single-stranded DNAs complementary to ADE17 and RPL4a/b RNA. Hybridization was detected by autoradiography and quantitated using AlphaImager 3400 software and shown as a percentage of wild-type expression. Expression of PHO5 - LacZ and RPL35a - LacZ fusion reporters were determined after growth of CY3003 and CY2706 in YPD. Assays were performed in triplicate
Figure Legend Snippet: Gene expression in the tra1-L3733A strain. a The promoter region of PHO5 was cloned as a LacZ reporter fusion into the LEU2 centromeric plasmid YCp87 and transformed into yeast strains CY4353 ( TRA1 ), CY4318 ( tra1 - A3727S ), CY4103 ( tra1 - L3733A ), CY4324 ( tra1 - F3740A ) and CY4350 ( tra1 - F3744A ), containing a plasmid copy of YHR100C. β-Galactosidase activity was determined after growth in low phosphate media for 15 h at 30°. Expression is shown as a percentage of that found for CY4353. Measurements were made in triplicate with the standard deviation indicated. b Expression from stress response elements. A cassette of two STRE elements was cloned into the Eco RI and Sac I sites of his3 - Δ88 - LacZ (Brandl et al. 1993 ) to give STRE - his3 - LacZ . These elements replace the normal Gcn4 binding site in HIS3 . STRE - his3 - LacZ and his3 - Δ88 were transformed into CY2706 and CY3003, and β-galactosidase assays performed after growth of cells in YPD containing 4% ethanol. c Expression levels determined by gene profiling (GP, mRNA-Seq) as compared with RNA dot blots ( ADE17 and RPL4a/b ) and LacZ reporter fusions ( PHO5 and RPL35a ). RNA was prepared from yeast strains CY3003 ( tra1 - L3733A ) and CY2706 ( TRA1 WT ). mRNA-Seq libraries were constructed and sequencing performed on the Illumina/Solexa Genome Analyzer II platform. Comparisons between the strains were made as outlined in “ Materials and methods ”. Similarly prepared RNA was spotted onto Hybond-N membrane and probed with single-stranded DNAs complementary to ADE17 and RPL4a/b RNA. Hybridization was detected by autoradiography and quantitated using AlphaImager 3400 software and shown as a percentage of wild-type expression. Expression of PHO5 - LacZ and RPL35a - LacZ fusion reporters were determined after growth of CY3003 and CY2706 in YPD. Assays were performed in triplicate

Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Transformation Assay, Activity Assay, Standard Deviation, Binding Assay, Construct, Sequencing, Hybridization, Autoradiography, Software

18) Product Images from "Characterization of a vacuolar sucrose transporter, HbSUT5, from Hevea brasiliensis: involvement in latex production through regulation of intracellular sucrose transport in the bark and laticifers"

Article Title: Characterization of a vacuolar sucrose transporter, HbSUT5, from Hevea brasiliensis: involvement in latex production through regulation of intracellular sucrose transport in the bark and laticifers

Journal: BMC Plant Biology

doi: 10.1186/s12870-019-2209-9

HbSUT5 as a single or low copy vacuolar  SUT  gene.  a . Phylogenetic tree of HbSUT5 and some selected plant sucrose transporters. Full length amino acid sequences of SUTs were exploited to create the phylogenetic tree by MEGA4. The GenBank accession numbers for the peptide sequences of other SUTs are At1g71880 (AtSUC1), At1g22710 (AtSUC2), At2g02860 (AtSUC3), At1g09960 (AtSUC4), At1g71890 (AtSUC5), At5g43610 (AtSUC6), At1g66570 (AtSUC7), At2g14670 (AtSUC8), At5g06170 (AtSUC9), BAA24071 (OsSUT1), BAC67163 (OsSUT2), BAB68368 (OsSUT3), BAC67164 (OsSUT4), BAC67165 (OsSUT5), ADW94615 (PtaSUT1), ADW94616 (PtaSUT3), ADW94617 (PtaSUT4), ADW94618 (PtaSUT5), ADW94619 (PtaSUT6), CAD61275 (LjSUT4), CAB75881 (HvSUT2), ABJ51933 (HbSUT1), ABJ51934 (HbSUT2A), ABJ51932 (HbSUT2B), ABK60190 (HbSUT3), ABK60191(HbSUT4) and ABK60189 (HbSUT5).  b-e . Localization of HbSUT5-GFP and OsTIP1;1-RFP in rice protoplasts (Scale bar = 5 μm).  b . Bright field image;  c . Transient expression of GFP;  d . Transient expression of RFP;  e . Merged GFP and RFP image.  f . DNA gel blot analysis of the HbSUT5 under high stringency. Genomic DNA from Hevea was digested with restriction analysis, resolved on a 0.8% agarose gel (20 μg/lane), and blotted to Hybond N + , and hybridized with a  32 P-labled 528-bp-long  HbSUT5  cDNA fragment
Figure Legend Snippet: HbSUT5 as a single or low copy vacuolar SUT gene. a . Phylogenetic tree of HbSUT5 and some selected plant sucrose transporters. Full length amino acid sequences of SUTs were exploited to create the phylogenetic tree by MEGA4. The GenBank accession numbers for the peptide sequences of other SUTs are At1g71880 (AtSUC1), At1g22710 (AtSUC2), At2g02860 (AtSUC3), At1g09960 (AtSUC4), At1g71890 (AtSUC5), At5g43610 (AtSUC6), At1g66570 (AtSUC7), At2g14670 (AtSUC8), At5g06170 (AtSUC9), BAA24071 (OsSUT1), BAC67163 (OsSUT2), BAB68368 (OsSUT3), BAC67164 (OsSUT4), BAC67165 (OsSUT5), ADW94615 (PtaSUT1), ADW94616 (PtaSUT3), ADW94617 (PtaSUT4), ADW94618 (PtaSUT5), ADW94619 (PtaSUT6), CAD61275 (LjSUT4), CAB75881 (HvSUT2), ABJ51933 (HbSUT1), ABJ51934 (HbSUT2A), ABJ51932 (HbSUT2B), ABK60190 (HbSUT3), ABK60191(HbSUT4) and ABK60189 (HbSUT5). b-e . Localization of HbSUT5-GFP and OsTIP1;1-RFP in rice protoplasts (Scale bar = 5 μm). b . Bright field image; c . Transient expression of GFP; d . Transient expression of RFP; e . Merged GFP and RFP image. f . DNA gel blot analysis of the HbSUT5 under high stringency. Genomic DNA from Hevea was digested with restriction analysis, resolved on a 0.8% agarose gel (20 μg/lane), and blotted to Hybond N + , and hybridized with a 32 P-labled 528-bp-long HbSUT5 cDNA fragment

Techniques Used: Expressing, Western Blot, Agarose Gel Electrophoresis

19) Product Images from "Two storage hexamerins from the beet armyworm Spodoptera exigua: Cloning, characterization and the effect of gene silencing on survival"

Article Title: Two storage hexamerins from the beet armyworm Spodoptera exigua: Cloning, characterization and the effect of gene silencing on survival

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-11-65

(A) SeHex and (B) SeSP1 transcript analysis by RT-PCR amplification after dsRNA injection . Three larval states (before death, still living and less vital) were randomly selected at each time point after injection. Total RNA was extracted and reversed to cDNA using AMV reverse transcriptase (Takara). SeHex and SeSP1 specific probes were radiolabeled with [α- 32 P]-dCTP. The specific primers SeHex-FP and SeHex-RP or SeSP1-FP and SeSP1-RP were used to amplify cDNAs in the same PCR reactions. The PCR products were separated on 2% agarose gel and transferred to a Hybond-N + nylon membrane. Hybridization, washing and signal detection of the blots were carried out as described previously. (A) Lanes marked 12 h (5L2), 24 h (5L3), 36 h (5L3), 48 h (W), 72 h (P1), 96 h (P2) and120 h (P3) indicate the times (and developmental stage) after injection. (B) Lanes marked 12 h (5L2), 24 h (5L3), 36 h (5L3), 48 h (W), 72 h (P1) and 96 h (P2) indicate the times (and developmental stage) after injection. The housekeeping gene β-actin was used as a reference.
Figure Legend Snippet: (A) SeHex and (B) SeSP1 transcript analysis by RT-PCR amplification after dsRNA injection . Three larval states (before death, still living and less vital) were randomly selected at each time point after injection. Total RNA was extracted and reversed to cDNA using AMV reverse transcriptase (Takara). SeHex and SeSP1 specific probes were radiolabeled with [α- 32 P]-dCTP. The specific primers SeHex-FP and SeHex-RP or SeSP1-FP and SeSP1-RP were used to amplify cDNAs in the same PCR reactions. The PCR products were separated on 2% agarose gel and transferred to a Hybond-N + nylon membrane. Hybridization, washing and signal detection of the blots were carried out as described previously. (A) Lanes marked 12 h (5L2), 24 h (5L3), 36 h (5L3), 48 h (W), 72 h (P1), 96 h (P2) and120 h (P3) indicate the times (and developmental stage) after injection. (B) Lanes marked 12 h (5L2), 24 h (5L3), 36 h (5L3), 48 h (W), 72 h (P1) and 96 h (P2) indicate the times (and developmental stage) after injection. The housekeeping gene β-actin was used as a reference.

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

20) Product Images from "Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus"

Article Title: Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Journal: Plant Physiology

doi:

Copy number of the ICS gene in C. roseus. DNA from leaves was digested with Eco RV (E) or Bam HI (B), separated, and blotted onto a Hybond-N + membrane. Hybridization was done with the full-length ICS cDNA.
Figure Legend Snippet: Copy number of the ICS gene in C. roseus. DNA from leaves was digested with Eco RV (E) or Bam HI (B), separated, and blotted onto a Hybond-N + membrane. Hybridization was done with the full-length ICS cDNA.

Techniques Used: Hybridization

21) Product Images from "Initiation of Protein Synthesis by Hepatitis C Virus Is Refractory to Reduced eIF2 ? GTP ? Met-tRNAiMet"

Article Title: Initiation of Protein Synthesis by Hepatitis C Virus Is Refractory to Reduced eIF2 ? GTP ? Met-tRNAiMet

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E06-06-0478

Analysis of initiation complexes formed on the HCV IRES in the presence of NSC119889. (A) Agarose gel analysis of RNA isolated from 48S and 80S complexes purified by HCV IRES chromatography. 48S and 80S complexes were formed on the HCV IRES as described in Materials and Methods in the presence of GMP-PNP or cycloheximide, respectively. RNA was isolated from these complexes, fractionated on a 1% agarose/formaldehyde, and visualized by staining with ethidium bromide. (B) Western blot detection of eIF2α and the p116 subunit of eIF3 in initiation complexes formed on the HCV IRES. Similar cell equivalents from the 48S and 80S complexes were TCA precipitated, fractionated by SDS-PAGE, and transferred to Immobilon P (Millipore) membrane. Immunoblots were performed with the indicated antibodies. The origin of each sample is indicated above the panel. (C) Detection of Met-tRNA i in the 48S and 80S initiation complexes. RNA was isolated from fractions containing 48S and 80S complexes using TRIzol, following the manufacturer's recommendation (Invitrogen). Five micrograms, representing 53 and 33% of the RNA recovered from 48S and 80S fractions, respectively, was analyzed on a 8 M urea/10% polyacrylamide gel and transferred onto a Hybond-N + membrane (GE Healthcare) using a Transblot SD semidry apparatus (Bio-Rad). The RNA was cross-linked using a UV-Stratalinker 2400 (Stratagene) and tRNA i Met detected by Northern blotting using a DNA oligonucleotide targeting mammalian tRNAi Met . (D) The HCV IRES uses Met-tRNA i recruited in the presence of NSC119889 for translation. Translations were performed in Krebs-2 extracts in the presence of unlabeled methionine and supplemented with in vitro charged [ 35 S]Met-tRNA i Met at the indicated concentrations of NSC119889. Translations were programmed with 10 μg/ml FF/HCV/Ren mRNA. Products were electrophoresed on a 10% SDS-polyacrylamide gel, treated with EN 3 Hance, dried, and exposed to X-OMAT film (Eastman Kodak).
Figure Legend Snippet: Analysis of initiation complexes formed on the HCV IRES in the presence of NSC119889. (A) Agarose gel analysis of RNA isolated from 48S and 80S complexes purified by HCV IRES chromatography. 48S and 80S complexes were formed on the HCV IRES as described in Materials and Methods in the presence of GMP-PNP or cycloheximide, respectively. RNA was isolated from these complexes, fractionated on a 1% agarose/formaldehyde, and visualized by staining with ethidium bromide. (B) Western blot detection of eIF2α and the p116 subunit of eIF3 in initiation complexes formed on the HCV IRES. Similar cell equivalents from the 48S and 80S complexes were TCA precipitated, fractionated by SDS-PAGE, and transferred to Immobilon P (Millipore) membrane. Immunoblots were performed with the indicated antibodies. The origin of each sample is indicated above the panel. (C) Detection of Met-tRNA i in the 48S and 80S initiation complexes. RNA was isolated from fractions containing 48S and 80S complexes using TRIzol, following the manufacturer's recommendation (Invitrogen). Five micrograms, representing 53 and 33% of the RNA recovered from 48S and 80S fractions, respectively, was analyzed on a 8 M urea/10% polyacrylamide gel and transferred onto a Hybond-N + membrane (GE Healthcare) using a Transblot SD semidry apparatus (Bio-Rad). The RNA was cross-linked using a UV-Stratalinker 2400 (Stratagene) and tRNA i Met detected by Northern blotting using a DNA oligonucleotide targeting mammalian tRNAi Met . (D) The HCV IRES uses Met-tRNA i recruited in the presence of NSC119889 for translation. Translations were performed in Krebs-2 extracts in the presence of unlabeled methionine and supplemented with in vitro charged [ 35 S]Met-tRNA i Met at the indicated concentrations of NSC119889. Translations were programmed with 10 μg/ml FF/HCV/Ren mRNA. Products were electrophoresed on a 10% SDS-polyacrylamide gel, treated with EN 3 Hance, dried, and exposed to X-OMAT film (Eastman Kodak).

Techniques Used: Agarose Gel Electrophoresis, Isolation, Purification, Chromatography, Staining, Western Blot, SDS Page, Northern Blot, In Vitro

22) Product Images from "Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1 [w]"

Article Title: Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1 [w]

Journal: Plant Physiology

doi: 10.1104/pp.104.056010

cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.
Figure Legend Snippet: cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.

Techniques Used: Hybridization, Labeling, Agarose Gel Electrophoresis, Molecular Weight

23) Product Images from "Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells"

Article Title: Heterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-4-18

Detection of small  lacZ  gene-specific RNA.  Total RNA was isolated from  Drosophila  cell cultures and fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels and stained with ethidium bromide before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. The membrane was probed with an antisense digoxigenin-labelled lacZ transcript corresponding to ~500 nt at the 5' end of the  lacZ  gene. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells (lanes 1, 2), DS2-HC-Pro cells (lanes 3, 4), DS2-2b cells (lanes 5, 6) and DS2-16k cells (lanes 7, 8). Cells represented in lanes 1, 3, 5 and 7 were transfected with pMT/V5-His/lacZ and those represented in lanes 2, 4, 6 and 8 were transfected with pMT/V5-His/lacZ and dsRNA. The line to the right indicates the position of GFP specific siRNAs produced in plants as described by Canto et al. (47). The lower panel shows the RNA species detected by staining of the gel before electroblotting.
Figure Legend Snippet: Detection of small lacZ gene-specific RNA. Total RNA was isolated from Drosophila cell cultures and fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels and stained with ethidium bromide before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. The membrane was probed with an antisense digoxigenin-labelled lacZ transcript corresponding to ~500 nt at the 5' end of the lacZ gene. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells (lanes 1, 2), DS2-HC-Pro cells (lanes 3, 4), DS2-2b cells (lanes 5, 6) and DS2-16k cells (lanes 7, 8). Cells represented in lanes 1, 3, 5 and 7 were transfected with pMT/V5-His/lacZ and those represented in lanes 2, 4, 6 and 8 were transfected with pMT/V5-His/lacZ and dsRNA. The line to the right indicates the position of GFP specific siRNAs produced in plants as described by Canto et al. (47). The lower panel shows the RNA species detected by staining of the gel before electroblotting.

Techniques Used: Isolation, Chromatography, Staining, Transfection, Produced

24) Product Images from "Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L)"

Article Title: Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L)

Journal: BMC Plant Biology

doi: 10.1186/1471-2229-3-6

Southern blot hybridization analyses of Ds insertion in the bfl1 mutant and Ds excision in revertants. Genomic DNA (~10 μg) was extracted from putative revertants, digested with Hind III, fractionated on a 0.7% agarose gel, and blotted to a Hybond-N + membrane. The membrane was hybridised with a gus probe (see Figure 6 ). PR1 to PR10 are revertants of the original bfl1 mutant and PR2-1 is one of the progenies of PR2. Abbreviations: N, wild-type phenotype; M, mutant phenotype.
Figure Legend Snippet: Southern blot hybridization analyses of Ds insertion in the bfl1 mutant and Ds excision in revertants. Genomic DNA (~10 μg) was extracted from putative revertants, digested with Hind III, fractionated on a 0.7% agarose gel, and blotted to a Hybond-N + membrane. The membrane was hybridised with a gus probe (see Figure 6 ). PR1 to PR10 are revertants of the original bfl1 mutant and PR2-1 is one of the progenies of PR2. Abbreviations: N, wild-type phenotype; M, mutant phenotype.

Techniques Used: Southern Blot, Hybridization, Mutagenesis, Agarose Gel Electrophoresis

25) Product Images from "Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms"

Article Title: Characterization of the Sesbania rostrata Phytochelatin Synthase Gene: Alternative Splicing and Function of Four Isoforms

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms10083269

Southern blot analysis of S. rostrata genomic DNA. S. rostrata genomic DNA (25μg) digested with Eco RI (lane 1), Bam HI (lane 2), Xba I (lane 3), and Pst I (lane 4) was separated by agarose gel electrophoresis, blotted onto Hybond-N membrane and hybridized to the 32 P-labeled conserved SrPCS cDNA fragment (nt 47-919 of GenBank GQ204309).
Figure Legend Snippet: Southern blot analysis of S. rostrata genomic DNA. S. rostrata genomic DNA (25μg) digested with Eco RI (lane 1), Bam HI (lane 2), Xba I (lane 3), and Pst I (lane 4) was separated by agarose gel electrophoresis, blotted onto Hybond-N membrane and hybridized to the 32 P-labeled conserved SrPCS cDNA fragment (nt 47-919 of GenBank GQ204309).

Techniques Used: Southern Blot, Agarose Gel Electrophoresis, Labeling

26) Product Images from "Tissue-Specific Processing of the Surf-5 and Surf-4 mRNAs"

Article Title: Tissue-Specific Processing of the Surf-5 and Surf-4 mRNAs

Journal: Gene Expression

doi:

Expression of the Surf-5b transcript in Neuro 2A cells. Poly(A) +  RNA (2  μ g) isolated from NIH 3T3 cells (A, lane 1), mouse brain (A, lane 2), or Neuro 2A cells (B) were separated on a 1% agarose gel containing formaldehyde, transferred to Hybond N, and probed with a full-length Surf-5 cDNA probe (A) or a Surf-5 coding region probe (B). The positions of the 3.5- and 1.5-kb transcripts are indicated.
Figure Legend Snippet: Expression of the Surf-5b transcript in Neuro 2A cells. Poly(A) + RNA (2 μ g) isolated from NIH 3T3 cells (A, lane 1), mouse brain (A, lane 2), or Neuro 2A cells (B) were separated on a 1% agarose gel containing formaldehyde, transferred to Hybond N, and probed with a full-length Surf-5 cDNA probe (A) or a Surf-5 coding region probe (B). The positions of the 3.5- and 1.5-kb transcripts are indicated.

Techniques Used: Expressing, Isolation, Agarose Gel Electrophoresis

Regulation of Surf-5b expression in differentiating P19 cells. (A) Total RNA from untreated (−) and day 8 retinoic acid-treated (+) P19 cells were separated on a 1% agarose gel containing formaldehyde, transferred to Hybond N, and probed with a 3′ UTR Surf-5 probe. The Surf-5 (3.5 kb mRNA) and the Surf-5b (1.5 kb) mRNAs are indicated. (B) Total cell lysates from untreated (–) and 8 day retinoic acid-treated (+) P19 cells were analyzed by Western blot analysis for the expression of Surf-5 proteins. The positions of the Surf-5 and Surf-5b proteins are indicated.
Figure Legend Snippet: Regulation of Surf-5b expression in differentiating P19 cells. (A) Total RNA from untreated (−) and day 8 retinoic acid-treated (+) P19 cells were separated on a 1% agarose gel containing formaldehyde, transferred to Hybond N, and probed with a 3′ UTR Surf-5 probe. The Surf-5 (3.5 kb mRNA) and the Surf-5b (1.5 kb) mRNAs are indicated. (B) Total cell lysates from untreated (–) and 8 day retinoic acid-treated (+) P19 cells were analyzed by Western blot analysis for the expression of Surf-5 proteins. The positions of the Surf-5 and Surf-5b proteins are indicated.

Techniques Used: Expressing, Agarose Gel Electrophoresis, Western Blot

27) Product Images from "Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells"

Article Title: Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl650

Analysis of the growth properties of wild type and Q74A mutant virus. ( a ) Plague sizes and one-step growth curves of rIBV and Q74A mutant viruses. Monolayers of Vero cells on a 6-well plate were infected with 100 µl of 1-, 10- and 100-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°C for 3 days. The cells were fixed and stained with 0.1% toluidine. To determine the one-step growth curves of wild-type recombinant IBV and Q74A mutant viruses, Vero cells were infected with wild type and Q74A mutant viruses, and harvested at 0, 8, 12, 16, 24, 36 and 48 h post-inoculation, respectively, and viral stocks were prepared by freezing/thawing of the cells three times. The viral titer was determined by plaque assay on Vero cells. ( b ) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with wild type and Q74A mutant viruses. Ten micrograms of total RNA extracted from Vero cells infected with rIBV and Q74A mutant viruses (passage 3 and 5), respectively, were separated on 1% agarose gel and transferred to a Hybond N+ membrane. Viral RNAs were probed with a Dig-labelled DNA probe corresponding to the 3-end 680 nt of the IBV genome. Total RNA extracted from mock-infected cells was included as negative control. Numbers on the left indicate nucleotides in kilobase and numbers on the right indicate the genomic and subgenomic RNA species of IBV. ( c ) Western blot analysis of viral protein expression in cells infected with wild type and Q74A mutant viruses. Vero cells infected with wild type recombinant IBV (lanes 1–7) and Q74A mutant virus (lanes 8–14) were harvested at 0, 8, 16, 24, 36 and 48 h postinfection, respectively, lysates prepared and separated on SDS-10% polyacrylamide gel. The expression of S and N proteins was analyzed by western blot with polyclonal anti-S and anti-N antibodies, respectively. The same membrane was also probed with anti-actin antibody as a loading control. Numbers on the left indicate molecular masses in kilodaltons. ( d ) Nucleotide sequencing of Q74A mutant virus. Total RNA was prepared from Vero cells infected with passage 5 of the Q74A mutant virus and the region covering the Q74A (CAA→GCA) mutation was amplified by RT–PCR and sequenced by automated nucleotide sequencing. A 30 nt region flanking the CAA→GCA mutation is shown.
Figure Legend Snippet: Analysis of the growth properties of wild type and Q74A mutant virus. ( a ) Plague sizes and one-step growth curves of rIBV and Q74A mutant viruses. Monolayers of Vero cells on a 6-well plate were infected with 100 µl of 1-, 10- and 100-fold diluted virus stock and cultured in the presence of 0.5% carboxymethy cellulose at 37°C for 3 days. The cells were fixed and stained with 0.1% toluidine. To determine the one-step growth curves of wild-type recombinant IBV and Q74A mutant viruses, Vero cells were infected with wild type and Q74A mutant viruses, and harvested at 0, 8, 12, 16, 24, 36 and 48 h post-inoculation, respectively, and viral stocks were prepared by freezing/thawing of the cells three times. The viral titer was determined by plaque assay on Vero cells. ( b ) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with wild type and Q74A mutant viruses. Ten micrograms of total RNA extracted from Vero cells infected with rIBV and Q74A mutant viruses (passage 3 and 5), respectively, were separated on 1% agarose gel and transferred to a Hybond N+ membrane. Viral RNAs were probed with a Dig-labelled DNA probe corresponding to the 3-end 680 nt of the IBV genome. Total RNA extracted from mock-infected cells was included as negative control. Numbers on the left indicate nucleotides in kilobase and numbers on the right indicate the genomic and subgenomic RNA species of IBV. ( c ) Western blot analysis of viral protein expression in cells infected with wild type and Q74A mutant viruses. Vero cells infected with wild type recombinant IBV (lanes 1–7) and Q74A mutant virus (lanes 8–14) were harvested at 0, 8, 16, 24, 36 and 48 h postinfection, respectively, lysates prepared and separated on SDS-10% polyacrylamide gel. The expression of S and N proteins was analyzed by western blot with polyclonal anti-S and anti-N antibodies, respectively. The same membrane was also probed with anti-actin antibody as a loading control. Numbers on the left indicate molecular masses in kilodaltons. ( d ) Nucleotide sequencing of Q74A mutant virus. Total RNA was prepared from Vero cells infected with passage 5 of the Q74A mutant virus and the region covering the Q74A (CAA→GCA) mutation was amplified by RT–PCR and sequenced by automated nucleotide sequencing. A 30 nt region flanking the CAA→GCA mutation is shown.

Techniques Used: Mutagenesis, Infection, Cell Culture, Staining, Recombinant, Plaque Assay, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Western Blot, Expressing, Sequencing, Cellular Antioxidant Activity Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

28) Product Images from "The Adsorption Protein Genes of Xanthomonas campestris Filamentous Phages Determining Host Specificity"

Article Title: The Adsorption Protein Genes of Xanthomonas campestris Filamentous Phages Determining Host Specificity

Journal: Journal of Bacteriology

doi:

Detection of hybrid phage released from nonpermissive host cells by spot test and Southern hybridization. (A) Five-microliter aliquots of the culture supernatants of P20H(pRKG3, φXvSG) and P20H(φXvSG) were separately spotted on a lawn of P20H(pRKG3), P20H, or Xv36, using the supernatants of P20H(φLf) and Xv36(φXv) as the controls. (B) The spotted regions on the lawn of P20H(pRKG3) (top row in panel A) were transferred by lifting onto a Hybond-N membrane and hybridized with the φLf gII or Gm r probe. The hybridization signal shown by the Xv36(φXv) supernatant (top row in panel B) was generated by the spotted phage φXv, which hybridized to the φLf gII , instead of progeny produced after spotting.
Figure Legend Snippet: Detection of hybrid phage released from nonpermissive host cells by spot test and Southern hybridization. (A) Five-microliter aliquots of the culture supernatants of P20H(pRKG3, φXvSG) and P20H(φXvSG) were separately spotted on a lawn of P20H(pRKG3), P20H, or Xv36, using the supernatants of P20H(φLf) and Xv36(φXv) as the controls. (B) The spotted regions on the lawn of P20H(pRKG3) (top row in panel A) were transferred by lifting onto a Hybond-N membrane and hybridized with the φLf gII or Gm r probe. The hybridization signal shown by the Xv36(φXv) supernatant (top row in panel B) was generated by the spotted phage φXv, which hybridized to the φLf gII , instead of progeny produced after spotting.

Techniques Used: Spot Test, Hybridization, Generated, Produced

29) Product Images from "Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase ▿"

Article Title: Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02490-09

Characterization of the growth properties and genetic stability of mutant viruses. (A) Growth properties of rIBV and mutant viruses. Vero cells were infected with wild-type and mutant viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, 24, and 36 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID 50 of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (B) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with wild-type and mutant viruses. Vero cells infected with wild-type and mutant viruses at an MOI of 1 PFU/cell. Total RNA (10 μg) extracted from the infected cells at 12 h postinfection was separated on 1% agarose gel and transferred to a Hybond N + membrane. Viral RNAs were probed with a Dig-labeled DNA probe corresponding to the 3′-end 400 nucleotides of the IBV genome. Numbers on the right indicate the genomic and subgenomic RNA species of IBV. (C) Partial reversion of Asn to Gln at amino acid position 3462 during passage of the Q3462N mutant virus. The plaque-purified Q3462N mutant virus was successively passaged on Vero cells using supernatants from the cells infected with this mutant virus at an MOI of 1 PFU/cell for 16 h. Total RNA was extracted from the infected cells, and RT-PCR was carried out. Sequencing the gel-purified RT-PCR products displayed an increasing trend reverting AAC to CAA, causing partial reversion of Asn to wild-type Gln from passage 2 to 5.
Figure Legend Snippet: Characterization of the growth properties and genetic stability of mutant viruses. (A) Growth properties of rIBV and mutant viruses. Vero cells were infected with wild-type and mutant viruses at an MOI of 1 PFU/cell and harvested at 0, 4, 8, 12, 16, 24, and 36 h postinoculation. Viral stocks were prepared by freeze-thawing the cells three times, and the TCID 50 of each viral stock was determined by infecting five wells of Vero cells on 96-well plates in triplicate with a 10-fold serial dilution of each viral stock. Error bar shows standard error of the mean. (B) Northern blot analysis of the genomic and subgenomic RNAs in cells infected with wild-type and mutant viruses. Vero cells infected with wild-type and mutant viruses at an MOI of 1 PFU/cell. Total RNA (10 μg) extracted from the infected cells at 12 h postinfection was separated on 1% agarose gel and transferred to a Hybond N + membrane. Viral RNAs were probed with a Dig-labeled DNA probe corresponding to the 3′-end 400 nucleotides of the IBV genome. Numbers on the right indicate the genomic and subgenomic RNA species of IBV. (C) Partial reversion of Asn to Gln at amino acid position 3462 during passage of the Q3462N mutant virus. The plaque-purified Q3462N mutant virus was successively passaged on Vero cells using supernatants from the cells infected with this mutant virus at an MOI of 1 PFU/cell for 16 h. Total RNA was extracted from the infected cells, and RT-PCR was carried out. Sequencing the gel-purified RT-PCR products displayed an increasing trend reverting AAC to CAA, causing partial reversion of Asn to wild-type Gln from passage 2 to 5.

Techniques Used: Mutagenesis, Infection, Serial Dilution, Northern Blot, Agarose Gel Electrophoresis, Labeling, Purification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Cellular Antioxidant Activity Assay

30) Product Images from "The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing"

Article Title: The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr044

Interaction of the 3′-hinge sequence of the U3 snoRNA with the 5′-ETS is required for SSU processome formation. Utp5-HA was immunoprecipitated with an anti-HA antibody from total cellular extract from yeast cells expressing the indicated U3 snoRNA mutants, at 16 h after the endogenous U3 snoRNA was depleted. ( A ) Total (5%) and immunoprecipitated proteins were separated by SDS–PAGE and transferred to Immobilon PVDF membranes. SSU processome assembly was assayed by western blotting for Mpp10 with an anti-Mpp10 antibody. Two different exposures are presented for the Mpp10 detection, a lighter (30 s) and a darker one (5 min). Formation of the UtpA/t-Utp subcomplex and its association with other components of the SSU processome was assayed by western blotting for Utp17-TAP with peroxidase-antiperoxidase antibodies (PAP), another UtpA/t-Utp subcomplex member. ( B ) The RNA extracted from either the total cellular extract (10%) or the immunoprecipitates was separated on denaturing polyacrylamide gels, transferred to Hybond N+ and the presence of U3 snoRNA was detected by northern blotting. U3 snoRNA expressed from a plasmid was detected with an oligonucleotide (SD13) that recognizes the unique sequence, while another oligonucleotide (SD74) was used to detect all forms of U3 snoRNA.
Figure Legend Snippet: Interaction of the 3′-hinge sequence of the U3 snoRNA with the 5′-ETS is required for SSU processome formation. Utp5-HA was immunoprecipitated with an anti-HA antibody from total cellular extract from yeast cells expressing the indicated U3 snoRNA mutants, at 16 h after the endogenous U3 snoRNA was depleted. ( A ) Total (5%) and immunoprecipitated proteins were separated by SDS–PAGE and transferred to Immobilon PVDF membranes. SSU processome assembly was assayed by western blotting for Mpp10 with an anti-Mpp10 antibody. Two different exposures are presented for the Mpp10 detection, a lighter (30 s) and a darker one (5 min). Formation of the UtpA/t-Utp subcomplex and its association with other components of the SSU processome was assayed by western blotting for Utp17-TAP with peroxidase-antiperoxidase antibodies (PAP), another UtpA/t-Utp subcomplex member. ( B ) The RNA extracted from either the total cellular extract (10%) or the immunoprecipitates was separated on denaturing polyacrylamide gels, transferred to Hybond N+ and the presence of U3 snoRNA was detected by northern blotting. U3 snoRNA expressed from a plasmid was detected with an oligonucleotide (SD13) that recognizes the unique sequence, while another oligonucleotide (SD74) was used to detect all forms of U3 snoRNA.

Techniques Used: Sequencing, Immunoprecipitation, Expressing, SDS Page, Western Blot, Northern Blot, Plasmid Preparation

31) Product Images from "Characterization of Influenza Virus NS1 Protein by Using a Novel Helper-Virus-Free Reverse Genetic System"

Article Title: Characterization of Influenza Virus NS1 Protein by Using a Novel Helper-Virus-Free Reverse Genetic System

Journal: Journal of Virology

doi:

Replication, transcription, and cytoplasmic accumulation of mRNAs. (A) Characterization of the vRNA. MDBK cells in a 10-cm-diameter dish were infected with the WSN, dl 12, or N110 virus at an MOI of 3, and the cells were incubated for the indicated times at 39°C. Poly(A) − RNAs were purified from the cells. RNAs were blotted onto a Hybond-N+ membrane and hybridized with plus-sense 32 P-labeled probe. (B) Characterization of total mRNA. MDBK cells were infected as described above and then incubated for the indicated times at 39°C. Poly(A) + RNAs were purified from the cells. RNAs were blotted onto a Hybond-N+ membrane and were hybridized with NP or M1 mRNA-specific minus-sense 32 P-labeled probe. (C) Characterization of cytoplasmic mRNA. MDBK cells were infected as described above and then incubated for the indicated times at 39°C. Poly(A) + RNAs were purified from the cytoplasmic fraction of the cells. RNAs were blotted onto a Hybond-N+ membrane and hybridized with NP or M1 mRNA-specific minus-sense 32 P-labeled probe. (D) The indicated amount of vRNAs, purified from the WSN virus, was used for blotting as a control. vRNA-specific plus-sense 32 P-labeled probe was used for hybridization.
Figure Legend Snippet: Replication, transcription, and cytoplasmic accumulation of mRNAs. (A) Characterization of the vRNA. MDBK cells in a 10-cm-diameter dish were infected with the WSN, dl 12, or N110 virus at an MOI of 3, and the cells were incubated for the indicated times at 39°C. Poly(A) − RNAs were purified from the cells. RNAs were blotted onto a Hybond-N+ membrane and hybridized with plus-sense 32 P-labeled probe. (B) Characterization of total mRNA. MDBK cells were infected as described above and then incubated for the indicated times at 39°C. Poly(A) + RNAs were purified from the cells. RNAs were blotted onto a Hybond-N+ membrane and were hybridized with NP or M1 mRNA-specific minus-sense 32 P-labeled probe. (C) Characterization of cytoplasmic mRNA. MDBK cells were infected as described above and then incubated for the indicated times at 39°C. Poly(A) + RNAs were purified from the cytoplasmic fraction of the cells. RNAs were blotted onto a Hybond-N+ membrane and hybridized with NP or M1 mRNA-specific minus-sense 32 P-labeled probe. (D) The indicated amount of vRNAs, purified from the WSN virus, was used for blotting as a control. vRNA-specific plus-sense 32 P-labeled probe was used for hybridization.

Techniques Used: Infection, Incubation, Purification, Labeling, Hybridization

32) Product Images from "A Novel Nuclear Member of the Thioredoxin Superfamily"

Article Title: A Novel Nuclear Member of the Thioredoxin Superfamily

Journal: Plant Physiology

doi:

RNA analysis of NRX gene expression. Approximately 7 μg of total RNA isolated from maize suspension-cultured cells, immature kernels, and from the leaves and roots of 1-week-old maize seedlings was size-fractionated in a 1.2% agarose Mops/formaldehyde gel. The RNA was then transferred to Hybond-N + membrane in 20× SSC. After UV cross-linking, the membrane was probed with the largest Eco RI insert fragment from ZmD1. This probe identified a transcript size of about 2 kb. The transcript appeared to be abundant in cultures as well as kernels. The mRNA was less abundant in leaves and roots.
Figure Legend Snippet: RNA analysis of NRX gene expression. Approximately 7 μg of total RNA isolated from maize suspension-cultured cells, immature kernels, and from the leaves and roots of 1-week-old maize seedlings was size-fractionated in a 1.2% agarose Mops/formaldehyde gel. The RNA was then transferred to Hybond-N + membrane in 20× SSC. After UV cross-linking, the membrane was probed with the largest Eco RI insert fragment from ZmD1. This probe identified a transcript size of about 2 kb. The transcript appeared to be abundant in cultures as well as kernels. The mRNA was less abundant in leaves and roots.

Techniques Used: Expressing, Isolation, Cell Culture

33) Product Images from "A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor ? gene expression: Molecular cloning, sequencing, characterization, and chromosomal assignment"

Article Title: A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor ? gene expression: Molecular cloning, sequencing, characterization, and chromosomal assignment

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

doi:

Northern blot hybridization of mRNA from THP-1 cells. The cells (2 × 10 7 ) were cultured in various conditions: stimulation with P. gingivalis LPS (LPS); differentiation with PMA (PMA); differentiation with PMA followed by stimulation with LPS (PMA+LPS); and no stimulation (plain). The mRNA was recovered from cells, run on denaturing formaldehyde/1.2% agarose gel, and transferred to a Hybond-N + filter. The filter was hybridized with the antisense RNA probe that corresponded to the coding region of the LITAF gene, as described in Materials and Methods . The hybridized filter was exposed to x-ray film for 24 hr. A 1.8-kb transcript was observed only in PMA+LPS-stimulated THP-1 cells. Similar amounts of β-actin mRNA were found in all mRNA tested (data not shown).
Figure Legend Snippet: Northern blot hybridization of mRNA from THP-1 cells. The cells (2 × 10 7 ) were cultured in various conditions: stimulation with P. gingivalis LPS (LPS); differentiation with PMA (PMA); differentiation with PMA followed by stimulation with LPS (PMA+LPS); and no stimulation (plain). The mRNA was recovered from cells, run on denaturing formaldehyde/1.2% agarose gel, and transferred to a Hybond-N + filter. The filter was hybridized with the antisense RNA probe that corresponded to the coding region of the LITAF gene, as described in Materials and Methods . The hybridized filter was exposed to x-ray film for 24 hr. A 1.8-kb transcript was observed only in PMA+LPS-stimulated THP-1 cells. Similar amounts of β-actin mRNA were found in all mRNA tested (data not shown).

Techniques Used: Northern Blot, Hybridization, Cell Culture, Agarose Gel Electrophoresis

34) Product Images from "Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines"

Article Title: Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601683

An example of a Northern blot for OPN and S100A4 mRNA levels in the clonal cell lines. The following samples were electrophoresed through 0.8% (w v −1 ) formaldehyde agarose gels and blotted onto Hybond-N membrane: Rama 800 (lane 1), R37C9Pooled (lane 2), R37C9VM13 (lane 3), R37C9VM16 (lane 4), R37C9VM8 (lane 5), R37C9VM6 (lane 6), R37C9VM25 (lane 7), Rama 37 (lane 8), R37C9VM29 (lane 9), R37C9VM14 (lane 10), R37C9VM3 (lane 11), R37C9VM18 (lane 12). In panel ( A ) the membrane was incubated with α 32 P-labelled OPN cDNA and subjected to autoradiography. In panel ( B ) the membrane was incubated with α 32 P-labelled S100A4 cDNA and subjected to autoradiography. In panel ( C ) the ethidium bromide-stained 18S and 28S ribosomal RNA bands on the membrane before hybridisation were photographed. The 1.6 kb OPN mRNA, 0.8 kb S100A4 mRNA and 18S and 28S ribosomal RNAs are shown. ( D ) Graph of OPN mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative OPN mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( E ) S100A4 mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative S100A4 mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( D , E ) Contain results for 9 C9-Met-DNA-transfected Rama 37 cell lines plus the uncloned C9-Met-DNA-transfected cells and the parental Rama 37 cells.
Figure Legend Snippet: An example of a Northern blot for OPN and S100A4 mRNA levels in the clonal cell lines. The following samples were electrophoresed through 0.8% (w v −1 ) formaldehyde agarose gels and blotted onto Hybond-N membrane: Rama 800 (lane 1), R37C9Pooled (lane 2), R37C9VM13 (lane 3), R37C9VM16 (lane 4), R37C9VM8 (lane 5), R37C9VM6 (lane 6), R37C9VM25 (lane 7), Rama 37 (lane 8), R37C9VM29 (lane 9), R37C9VM14 (lane 10), R37C9VM3 (lane 11), R37C9VM18 (lane 12). In panel ( A ) the membrane was incubated with α 32 P-labelled OPN cDNA and subjected to autoradiography. In panel ( B ) the membrane was incubated with α 32 P-labelled S100A4 cDNA and subjected to autoradiography. In panel ( C ) the ethidium bromide-stained 18S and 28S ribosomal RNA bands on the membrane before hybridisation were photographed. The 1.6 kb OPN mRNA, 0.8 kb S100A4 mRNA and 18S and 28S ribosomal RNAs are shown. ( D ) Graph of OPN mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative OPN mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( E ) S100A4 mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative S100A4 mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). ( D , E ) Contain results for 9 C9-Met-DNA-transfected Rama 37 cell lines plus the uncloned C9-Met-DNA-transfected cells and the parental Rama 37 cells.

Techniques Used: Northern Blot, Incubation, Autoradiography, Staining, Hybridization, Clone Assay, In Vivo, Transfection

An example of Southern hybridisation of C9-Met-DNA to DNA obtained from the clonal cell lines. ( A ) DNA from each of the cell lines was digested with Eco R1, electrophoresed on agarose gels, and blotted onto Hybond-N membrane. The samples loaded on the gel were 500 copies C9-Met-DNA (lane 1), 50 copies C9-Met-DNA (lane 2), Rama 37 DNA (lane 3), R37C9VM18 (lane 4), R37C9VM13 (lane 5), R37C9VM6 (lane 6), R37C9VM29 (lane 7), R37C9VM14 (lane 8), R37C9VM3 (lane 9), R37C9VM8 (lane 10), R37C9VM16 (lane 11) and R37C9VM25 (lane 12). The membrane was incubated with α 32 P-labelled C9-Met-DNA under hybridising conditions and subjected to autoradiography. The position of the authentic C9-Met-DNA is shown by the arrow. ( B ) Graph of C9-Met-DNA copy number (copy no) of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). Results for the nine cloned C9-Met-DNA-transfected cell lines plus Rama 37 parental cells are shown.
Figure Legend Snippet: An example of Southern hybridisation of C9-Met-DNA to DNA obtained from the clonal cell lines. ( A ) DNA from each of the cell lines was digested with Eco R1, electrophoresed on agarose gels, and blotted onto Hybond-N membrane. The samples loaded on the gel were 500 copies C9-Met-DNA (lane 1), 50 copies C9-Met-DNA (lane 2), Rama 37 DNA (lane 3), R37C9VM18 (lane 4), R37C9VM13 (lane 5), R37C9VM6 (lane 6), R37C9VM29 (lane 7), R37C9VM14 (lane 8), R37C9VM3 (lane 9), R37C9VM8 (lane 10), R37C9VM16 (lane 11) and R37C9VM25 (lane 12). The membrane was incubated with α 32 P-labelled C9-Met-DNA under hybridising conditions and subjected to autoradiography. The position of the authentic C9-Met-DNA is shown by the arrow. ( B ) Graph of C9-Met-DNA copy number (copy no) of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). Results for the nine cloned C9-Met-DNA-transfected cell lines plus Rama 37 parental cells are shown.

Techniques Used: Hybridization, Incubation, Autoradiography, In Vivo, Clone Assay, Transfection

35) Product Images from "Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines"

Article Title: Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0703539

Northern blot analysis of iNOS expression in cell lines transfected with pSG5/iNOS. RNA was electrophoresed through a 1.5% agarose gel, transferred to Hybond N+ membranes and hybridized with  32 P-labelled double-stranded probe to murine iNOS (includes the region used for sense/antisense). Cells were either unstimulated or stimulated for 4 h with LPS/IFN-γ (1 μg ml −1  LPS, 50 u ml −1  IFN-γ, 25 ml −1  175 cm 3  flask 2 × 10 7  cells −1 ). The bands of antisense or sense sequence plus β-globin from pSG5 are approximately 1.2 kb. The band for endogenous iNOS mRNA (in the stimulated sEnd-1 and S7 but not visible in A1) is approximately 3.6 kb. Lane 1: stimulated S7; lane 2: unstimulated S7; lane 3: stimulated A1; lane 4: unstimulated A1; lane 5: stimulated untransfected sEnd-1; lane 6: unstimulated untransfected sEnd-1.
Figure Legend Snippet: Northern blot analysis of iNOS expression in cell lines transfected with pSG5/iNOS. RNA was electrophoresed through a 1.5% agarose gel, transferred to Hybond N+ membranes and hybridized with 32 P-labelled double-stranded probe to murine iNOS (includes the region used for sense/antisense). Cells were either unstimulated or stimulated for 4 h with LPS/IFN-γ (1 μg ml −1 LPS, 50 u ml −1 IFN-γ, 25 ml −1 175 cm 3 flask 2 × 10 7 cells −1 ). The bands of antisense or sense sequence plus β-globin from pSG5 are approximately 1.2 kb. The band for endogenous iNOS mRNA (in the stimulated sEnd-1 and S7 but not visible in A1) is approximately 3.6 kb. Lane 1: stimulated S7; lane 2: unstimulated S7; lane 3: stimulated A1; lane 4: unstimulated A1; lane 5: stimulated untransfected sEnd-1; lane 6: unstimulated untransfected sEnd-1.

Techniques Used: Northern Blot, Expressing, Transfection, Agarose Gel Electrophoresis, Sequencing

36) Product Images from "Genetic Analysis of Borrelia garinii OspA Serotype 4 Strains Associated with Neuroborreliosis: Evidence for Extensive Genetic Homogeneity"

Article Title: Genetic Analysis of Borrelia garinii OspA Serotype 4 Strains Associated with Neuroborreliosis: Evidence for Extensive Genetic Homogeneity

Journal: Journal of Clinical Microbiology

doi:

RFLP pattern analysis of the UHB element. DNA isolated from each strain was digested with Hae III and was fractionated in a 0.8% GTG agarose gel. The DNA was then transferred to Hybond-N membranes and was hybridized with the 5′-end-labeled uhb(+) oligonucleotide probe. All procedures are described in the text. Size standards (in kilobases) are indicated on the right.
Figure Legend Snippet: RFLP pattern analysis of the UHB element. DNA isolated from each strain was digested with Hae III and was fractionated in a 0.8% GTG agarose gel. The DNA was then transferred to Hybond-N membranes and was hybridized with the 5′-end-labeled uhb(+) oligonucleotide probe. All procedures are described in the text. Size standards (in kilobases) are indicated on the right.

Techniques Used: Isolation, Agarose Gel Electrophoresis, Labeling

37) Product Images from "Identification and Differential Expression of Two Thioredoxin h Isoforms in Germinating Seeds from Pea 1"

Article Title: Identification and Differential Expression of Two Thioredoxin h Isoforms in Germinating Seeds from Pea 1

Journal: Plant Physiology

doi: 10.1104/pp.102.019562

Expression of Trx h isoforms in seeds and seedlings on northern blots. Total RNA from cotyledons and embryo axes from mature seeds imbibed for 0 to 46 h (UG, ungerminated seeds; G, germinated seeds) and from green leaves (GL) and roots (R) from 7-d-old seedlings were resolved per lane of 1.4% (w/v) agarose denaturing gels. They were transferred onto Hybond N membrane and probed with [ 32 P]-labeled coding regions of Trx h1 to h4 cDNA or [ 32 P]-labeled 18 S cDNA from Medicago truncatula . After hybridization and stringent washing (0.2× SSC and 0.02% [w/v] SDS at 65°C), membranes were autoradiographied for 3 d at –80°C.
Figure Legend Snippet: Expression of Trx h isoforms in seeds and seedlings on northern blots. Total RNA from cotyledons and embryo axes from mature seeds imbibed for 0 to 46 h (UG, ungerminated seeds; G, germinated seeds) and from green leaves (GL) and roots (R) from 7-d-old seedlings were resolved per lane of 1.4% (w/v) agarose denaturing gels. They were transferred onto Hybond N membrane and probed with [ 32 P]-labeled coding regions of Trx h1 to h4 cDNA or [ 32 P]-labeled 18 S cDNA from Medicago truncatula . After hybridization and stringent washing (0.2× SSC and 0.02% [w/v] SDS at 65°C), membranes were autoradiographied for 3 d at –80°C.

Techniques Used: Expressing, Northern Blot, Labeling, Hybridization

38) Product Images from "Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis"

Article Title: Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

Journal: Journal of Proteome Research

doi: 10.1021/acs.jproteome.6b00717

Outline of SILAC analysis. (A) CCRF-CEM cells were labeled with heavy or light arginine and lysine isotopes. Half the cells were treated with 0.9 μM 6-TG for 24 h and the other half left untreated. Half of each of these two cultures was then irradiated with 50 kJ/m 2 UVA. The remaining four cultures were not irradiated. Cells were mixed in 1:1 ratios for preparation of chromatin or whole cell extracts (RIPA) as indicated. Chromatin extracts were applied to a HyBond-N + membrane that was water and 8 M urea washed prior to in situ trypsin digestion and MS analysis. RIPA extracts were subjected to short SDS-PAGE and in-gel trypsin digestion prior to MS analysis. (B) The 16 mixes generated from different combinations of treatments.
Figure Legend Snippet: Outline of SILAC analysis. (A) CCRF-CEM cells were labeled with heavy or light arginine and lysine isotopes. Half the cells were treated with 0.9 μM 6-TG for 24 h and the other half left untreated. Half of each of these two cultures was then irradiated with 50 kJ/m 2 UVA. The remaining four cultures were not irradiated. Cells were mixed in 1:1 ratios for preparation of chromatin or whole cell extracts (RIPA) as indicated. Chromatin extracts were applied to a HyBond-N + membrane that was water and 8 M urea washed prior to in situ trypsin digestion and MS analysis. RIPA extracts were subjected to short SDS-PAGE and in-gel trypsin digestion prior to MS analysis. (B) The 16 mixes generated from different combinations of treatments.

Techniques Used: Labeling, Irradiation, In Situ, Mass Spectrometry, SDS Page, Generated

Cross-linking by ciprofloxacin+UVA. (A) Outline of analysis. Heavy- or light-labeled CCRF-CEM cells were treated with 500 μM ciprofloxacin (Cip) for 1 h and UVA (50 kJ/m 2 ) as indicated. Salt-washed chromatin prepared from cells mixed in 1:1 ratios as indicated was applied to a HyBond-N + membrane. Following washing with water and 8 M urea, membrane-associated proteins were trypsin digested in situ and analyzed by MS. The table describes the mixes that were compared. (B) Ciprofloxacin+UVA induced DPC formation. Scatter plot of Mix 2 versus Mix 3. (C) DNA and RNA binding proteins among Cirofloxacin+UVA (green) and 6-TG+UVA (blue). (D) Cross-linking profiles for 29 DNA repair proteins that best fit the most vulnerable profile. (E) Overlap between 29 DNA repair proteins cross-linked by ciprofloxacin+UVA and the 30 cross-linked by 6-TG+UVA.
Figure Legend Snippet: Cross-linking by ciprofloxacin+UVA. (A) Outline of analysis. Heavy- or light-labeled CCRF-CEM cells were treated with 500 μM ciprofloxacin (Cip) for 1 h and UVA (50 kJ/m 2 ) as indicated. Salt-washed chromatin prepared from cells mixed in 1:1 ratios as indicated was applied to a HyBond-N + membrane. Following washing with water and 8 M urea, membrane-associated proteins were trypsin digested in situ and analyzed by MS. The table describes the mixes that were compared. (B) Ciprofloxacin+UVA induced DPC formation. Scatter plot of Mix 2 versus Mix 3. (C) DNA and RNA binding proteins among Cirofloxacin+UVA (green) and 6-TG+UVA (blue). (D) Cross-linking profiles for 29 DNA repair proteins that best fit the most vulnerable profile. (E) Overlap between 29 DNA repair proteins cross-linked by ciprofloxacin+UVA and the 30 cross-linked by 6-TG+UVA.

Techniques Used: Labeling, In Situ, Mass Spectrometry, RNA Binding Assay

HyBond-N + membrane binding of DNA cross-linked proteins. (A) Sheared, salt-washed chromatin from CCRF-CEM cells that were untreated or treated with 6-TG and 50 kJ/m 2 UVA as indicated was applied to HyBond-N + membranes. Membranes were washed extensively with water and 8 M urea as indicated. Membrane-associated protein and DNA was visualized by staining with Sypro Ruby and SYBR Green. XPA protein was detected by immunostaining. (B) DNA-bound protein. Sheared chromatin from untreated CCRF-CEM cells or cells treated with UVA, 6-TG or 6-TG+UVA was applied to HyBond-N + membranes. Following extensive washing with water and 8 M urea, membranes were probed with antibodies as indicated.
Figure Legend Snippet: HyBond-N + membrane binding of DNA cross-linked proteins. (A) Sheared, salt-washed chromatin from CCRF-CEM cells that were untreated or treated with 6-TG and 50 kJ/m 2 UVA as indicated was applied to HyBond-N + membranes. Membranes were washed extensively with water and 8 M urea as indicated. Membrane-associated protein and DNA was visualized by staining with Sypro Ruby and SYBR Green. XPA protein was detected by immunostaining. (B) DNA-bound protein. Sheared chromatin from untreated CCRF-CEM cells or cells treated with UVA, 6-TG or 6-TG+UVA was applied to HyBond-N + membranes. Following extensive washing with water and 8 M urea, membranes were probed with antibodies as indicated.

Techniques Used: Binding Assay, Staining, SYBR Green Assay, Immunostaining

39) Product Images from "Cloning and Functional Expression of an Aplysia 5-HT Receptor Negatively Coupled to Adenylate Cyclase"

Article Title: Cloning and Functional Expression of an Aplysia 5-HT Receptor Negatively Coupled to Adenylate Cyclase

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.18-15-05586.1998

Southern blot analysis of the 5-HT ap1 gene. Genomic DNA was isolated from ovotestis, purified, and digested with  Bam HI ( lane 1 ),  Bgl II ( lane 2 ),  Eco RI ( lane 3 ), Hin dIII ( lane 4 ), Sac I ( lane 5 ), or  Xba I ( lane 6 ). Digested DNA was run on a 1% agarose gel, transferred to a Hybond N+ membrane, and hybridized with a 32 P-labeled 5-HTap.B cDNA fragment (nt 457–1927). Bgl II,  Eco RI,  Sac I, and Xba I sites are not present in the probe sequence, whereas  Bam HI and  Hin dIII appear once. The molecular weight marker is λ phage DNA digested with Hin dIII. The molecular weights are indicated in kilobase pairs.
Figure Legend Snippet: Southern blot analysis of the 5-HT ap1 gene. Genomic DNA was isolated from ovotestis, purified, and digested with Bam HI ( lane 1 ), Bgl II ( lane 2 ), Eco RI ( lane 3 ), Hin dIII ( lane 4 ), Sac I ( lane 5 ), or Xba I ( lane 6 ). Digested DNA was run on a 1% agarose gel, transferred to a Hybond N+ membrane, and hybridized with a 32 P-labeled 5-HTap.B cDNA fragment (nt 457–1927). Bgl II, Eco RI, Sac I, and Xba I sites are not present in the probe sequence, whereas Bam HI and Hin dIII appear once. The molecular weight marker is λ phage DNA digested with Hin dIII. The molecular weights are indicated in kilobase pairs.

Techniques Used: Southern Blot, Isolation, Purification, Agarose Gel Electrophoresis, Labeling, Sequencing, Molecular Weight, Marker

40) Product Images from "Embryonic Activation and Developmental Expression of the Murine Prion Protein Gene"

Article Title: Embryonic Activation and Developmental Expression of the Murine Prion Protein Gene

Journal: Gene Expression

doi:

PrP RNA expression in postnatal and adult tissues. Total RNA was prepared from brains collected at 1 (P1), 10 (P10), 20 (P20), and 42 (P42) days of postnatal development (pooled from six animals), and from adult (P42) heart (H), lung (Lg), testis, (T), kidney (K), spleen (S), liver, (Lv), and skeletal muscle (M) (pooled from four animals). Denatured RNA (20 μg) was size fractionated through a 1% agarose gel, containing formaldehyde, transferred to Hybond ® -N, and the membrane hybridized with a radiolabeled PrP probe (upper panel) and exposed to BiomaxMS autoradiography film (Sigma, Dorset, UK) for 24 h. Membranes were stripped and reprobed with an 18S rRNA cDNA probe (lower panel) to normalize for variations in RNA loading. The positions of 28S (4712 nt) and 18S (1869 nt) rRNA are indicated. (a) Expression of PrP in postnatal developing brain and various adult tissues. (b) Expression of PrP in various  Prnp + / +  and  Prnp − / −  tissues. (c) PrP phosphorimager values were adjusted in relation to 18S values and expressed as a percentage of PrP expression in the P42 adult brain. Shown are the means and SDs of values from three separate Northern blots. PrP RNA expression values are presented relative to values obtained for P42 brain.
Figure Legend Snippet: PrP RNA expression in postnatal and adult tissues. Total RNA was prepared from brains collected at 1 (P1), 10 (P10), 20 (P20), and 42 (P42) days of postnatal development (pooled from six animals), and from adult (P42) heart (H), lung (Lg), testis, (T), kidney (K), spleen (S), liver, (Lv), and skeletal muscle (M) (pooled from four animals). Denatured RNA (20 μg) was size fractionated through a 1% agarose gel, containing formaldehyde, transferred to Hybond ® -N, and the membrane hybridized with a radiolabeled PrP probe (upper panel) and exposed to BiomaxMS autoradiography film (Sigma, Dorset, UK) for 24 h. Membranes were stripped and reprobed with an 18S rRNA cDNA probe (lower panel) to normalize for variations in RNA loading. The positions of 28S (4712 nt) and 18S (1869 nt) rRNA are indicated. (a) Expression of PrP in postnatal developing brain and various adult tissues. (b) Expression of PrP in various Prnp + / + and Prnp − / − tissues. (c) PrP phosphorimager values were adjusted in relation to 18S values and expressed as a percentage of PrP expression in the P42 adult brain. Shown are the means and SDs of values from three separate Northern blots. PrP RNA expression values are presented relative to values obtained for P42 brain.

Techniques Used: RNA Expression, Agarose Gel Electrophoresis, Autoradiography, Expressing, Northern Blot

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Agarose Gel Electrophoresis:

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Article Snippet: .. Total RNA (20 μg) or DNA (2 μg) was subjected to electrophoresis in an agarose-formaldehyde or agarose gel and blotted onto Hybond-N+ membrane (Amersham Pharmacia). .. To make antisense or sense RNA probe, the plasmid pAtHD1–2 was linearized by digestion with Xho I or Xba I, respectively, and was used as a template in separate reactions to generate single-stranded RNA with in vitro synthesis kits (Promega).

Article Title: Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines
Article Snippet: .. RNA for Northern blotting was resolved on a 0.8% (w v−1 ) formaldehyde agarose gel and blotted onto Hybond-N membrane (Amersham Pharmacia) in 10 × SSC. ..

Northern Blot:

Article Title: Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines
Article Snippet: .. RNA for Northern blotting was resolved on a 0.8% (w v−1 ) formaldehyde agarose gel and blotted onto Hybond-N membrane (Amersham Pharmacia) in 10 × SSC. ..

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION
Article Snippet: .. For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). .. Prehybridization (30 min) and hybridization (3 h) were performed at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA).

Labeling:

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION
Article Snippet: .. For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). .. Prehybridization (30 min) and hybridization (3 h) were performed at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA).

Electrophoresis:

Article Title: Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development
Article Snippet: .. Total RNA (20 μg) or DNA (2 μg) was subjected to electrophoresis in an agarose-formaldehyde or agarose gel and blotted onto Hybond-N+ membrane (Amersham Pharmacia). .. To make antisense or sense RNA probe, the plasmid pAtHD1–2 was linearized by digestion with Xho I or Xba I, respectively, and was used as a template in separate reactions to generate single-stranded RNA with in vitro synthesis kits (Promega).

Article Title: Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide
Article Snippet: .. Samples were then subjected to electrophoresis through 1.5% (wv−1 ) agarose-formaldehyde gels and transferred to a Hybond-N+ membrane (Amersham Life Sciences, U.K.) according to the manufacturer's instructions. .. Transfer was by diffusion overnight with 20× SSPE as buffer (3.6 M sodium chloride, 0.2 M sodium phosphate (pH 7.7) and 0.02 M EDTA).

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION
Article Snippet: .. For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). .. Prehybridization (30 min) and hybridization (3 h) were performed at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA).

DNA Labeling:

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION
Article Snippet: .. For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). .. Prehybridization (30 min) and hybridization (3 h) were performed at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA).

Hybridization:

Article Title: GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION
Article Snippet: .. For Northern blot hybridization, 15 μg of total RNA was resolved on 1% agarose/formaldehyde gels by electrophoresis, transferred to Hybond=N+ membrane (Amersham Pharmacia Biotech, Buckinhamshire, England), aprobed with IL-8 cDNA labeled with [α-32 P] dATP (Perkin-Elmer Life Sciences Inc., Boston, MA) using a random-priming DNA-labeling kit (Stratagene, Cedar Creek, TX). .. Prehybridization (30 min) and hybridization (3 h) were performed at 68°C in Express Hybridization solution (Clontech, Palo Alto, CA).

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    GE Healthcare hybond n nylon membranes
    Northern blot analyses of selected ESTs under ABA treatment. Total RNA was denatured, transferred to <t>Hybond-N</t> + nylon membranes, and hybridized with DIG-labeled probes. LOX, lipoxygenase; HPP, hydroxyphenyl pyruvate integral membrane protein; XTH, xyloglucan endotransglucosylase/hydrolase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase; C, untreated control; 6 h, 12 h and 24 h indicate hours of ABA treatment.
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    Detection of FTD by anti-BrdU antibodies. ( A ) Dot blot analysis of chemically synthesized FTD. BSA-conjugated FTD or non-conjugated (NC) BSA on nitrocellulose membrane were blotted with anti-BrdU antibodies. ( B ) Dot blot analysis of FTD or BrdU incorporated into the genomic DNA of HCT-116 cells. HCT-116 cells were cultured in the presence of 5 μM FTD or BrdU for 4 hours and genomic DNA was purified. Purified DNA was denatured with alkaline solution (0.1 N NaOH), spotted onto <t>Hybond-N</t> + membrane and blotted with anti-BrdU antibodies.
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    Southern blot and PCR analyses of M. smegmatis LS2 strains. The genomic DNA was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to <t>Hybond-N</t> + membrane and hybridized by MSMEG_1556 probe. A. The expected DNA fragments hybridized by MSMEG_1556 probe were 2.12 kb and 2.55 kb. The expected size of PCR product from M. smegmatis LS2 strain was 3.15 kb. B. The expected DNA fragments hybridized by MSMEG_1556 probe was 3.37 kb and the expected size of PCR product from M. smegmatis mc 2 155 strain was 1.88 kb. C. Confirmation of M. smegmatis LS2 strains by Southern blot. Lanes 1–5. M. smegmatis LS2 strains have the hybridized DNA bands of 2.12 kb and 2.55 kb; lane 6. wild type M. smegmatis mc 2 155 has the hybridized DNA band of 3.37 kb. D. Confirmation of M. smegmatis LS2 strains by PCR. Lanes 1–5. the PCR product (3.15 kb) from M. smegmatis LS2 strains; lane 6. the PCR product (1.88 kb) from wild type M. smegmatis mc 2 155.
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    Northern blot analyses of selected ESTs under ABA treatment. Total RNA was denatured, transferred to Hybond-N + nylon membranes, and hybridized with DIG-labeled probes. LOX, lipoxygenase; HPP, hydroxyphenyl pyruvate integral membrane protein; XTH, xyloglucan endotransglucosylase/hydrolase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase; C, untreated control; 6 h, 12 h and 24 h indicate hours of ABA treatment.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Northern blot analyses of selected ESTs under ABA treatment. Total RNA was denatured, transferred to Hybond-N + nylon membranes, and hybridized with DIG-labeled probes. LOX, lipoxygenase; HPP, hydroxyphenyl pyruvate integral membrane protein; XTH, xyloglucan endotransglucosylase/hydrolase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase; C, untreated control; 6 h, 12 h and 24 h indicate hours of ABA treatment.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Northern Blot, Labeling

    Expression analyses of selected ESTs under osmotic stress. (A) Northern blot analysis. Total RNA was denatured, transferred to Hybond-N + nylon membranes and hybridized with DIG-labeled probes. (B) Expression analysis by RT-PCR. LEA, late embryogenesis abundant family protein; PEAMT, phosphoethanolamine methyltransferase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; XTH, xyloglucan endotransglucosylase/hydrolase; DHN, dehydrin; LOX, lipoxygenase; FNR, ferredoxin-NADP(H) oxidoreductase. C, unstressed control; 1 d, 2 d, 3 d, 5 d and 6 d indicate days of osmotic stress.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Expression analyses of selected ESTs under osmotic stress. (A) Northern blot analysis. Total RNA was denatured, transferred to Hybond-N + nylon membranes and hybridized with DIG-labeled probes. (B) Expression analysis by RT-PCR. LEA, late embryogenesis abundant family protein; PEAMT, phosphoethanolamine methyltransferase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; XTH, xyloglucan endotransglucosylase/hydrolase; DHN, dehydrin; LOX, lipoxygenase; FNR, ferredoxin-NADP(H) oxidoreductase. C, unstressed control; 1 d, 2 d, 3 d, 5 d and 6 d indicate days of osmotic stress.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction

    Confirmation of differential expression of clones from the osmotic stress SSH cDNA library. cDNAs from the forward subtraction were randomly picked and used to prepare two identical Hybond-N + nylon membranes. Clones were hybridized with (A) forward- or (B) reverse-subtracted probes.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Confirmation of differential expression of clones from the osmotic stress SSH cDNA library. cDNAs from the forward subtraction were randomly picked and used to prepare two identical Hybond-N + nylon membranes. Clones were hybridized with (A) forward- or (B) reverse-subtracted probes.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Expressing, Clone Assay, cDNA Library Assay

    Estimation of the copy number of genes for (A) ARF, (B) CIP and (C) PEAMT by Southern blot analysis. DNA was digested, electrophoresed, denatured, neutralized, transferred to Hybond-N + nylon membrane and hybridized with DIG-labeled probes. ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Estimation of the copy number of genes for (A) ARF, (B) CIP and (C) PEAMT by Southern blot analysis. DNA was digested, electrophoresed, denatured, neutralized, transferred to Hybond-N + nylon membrane and hybridized with DIG-labeled probes. ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Southern Blot, Labeling

    Noncoding RNAs connected with the flv4-2 operon. A , schematic presentation of chromosomal arrangement of the genes flv4 , sll 0218, and flv2 in the flv4-2 operon; ctpB ; the antisense transcribed sections for as1_flv4 , as2_flv4 , and as_flv2 ; and the gene for ncRNA, ncr0080. B , verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M , molecular mass marker; R , total RNA after electrophoresis. C , classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface , and the transcription start for flv4 , according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow . Predicted −10 and −35 elements are underlined . The chromosomal positions are according to Cyanobase.

    Journal: The Journal of Biological Chemistry

    Article Title: The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply *

    doi: 10.1074/jbc.M112.391755

    Figure Lengend Snippet: Noncoding RNAs connected with the flv4-2 operon. A , schematic presentation of chromosomal arrangement of the genes flv4 , sll 0218, and flv2 in the flv4-2 operon; ctpB ; the antisense transcribed sections for as1_flv4 , as2_flv4 , and as_flv2 ; and the gene for ncRNA, ncr0080. B , verification of transcript accumulation by Northern blot analysis. RNA was isolated from HC-grown Synechocystis WT cells, separated by denaturing electrophoresis, and blotted onto a Hybond-N nylon membrane. RNA was stained with methylene blue. Specific radiolabeled probes were derived from in vitro transcription. Arrows indicate the selected signals. M , molecular mass marker; R , total RNA after electrophoresis. C , classification of the noncoding RNA Ncr0080. Shown is the region in between the start codons for ctpB (168533–168535) and flv4 (168152–168150). The ncr0080 sequence is in gray boldface , and the transcription start for flv4 , according to the results of 5′-rapid amplification of cDNA end experiments, is marked with an arrow . Predicted −10 and −35 elements are underlined . The chromosomal positions are according to Cyanobase.

    Article Snippet: To characterize small RNAs, RNA samples (5 μg) were mixed with RNA loading dye (Fermentas, St. Leon-Rot, Germany), denatured for 10 min at 70 °C, separated in 10% polyacrylamide-urea gels, and transferred to Hybond-N nylon membranes (GE Healthcare) by electroblotting for 1 h. For the mRNA studies, RNA samples were mixed with denaturation solution, incubated for 10 min at 70 °C, separated in 1.3% agarose gels containing 7% formaldehyde in MOPS, and transferred to Hybond-N nylon membranes by capillary blotting with 10× SSC overnight ( ).

    Techniques: Northern Blot, Isolation, Electrophoresis, Staining, Derivative Assay, In Vitro, Marker, Sequencing, Amplification

    Detection of FTD by anti-BrdU antibodies. ( A ) Dot blot analysis of chemically synthesized FTD. BSA-conjugated FTD or non-conjugated (NC) BSA on nitrocellulose membrane were blotted with anti-BrdU antibodies. ( B ) Dot blot analysis of FTD or BrdU incorporated into the genomic DNA of HCT-116 cells. HCT-116 cells were cultured in the presence of 5 μM FTD or BrdU for 4 hours and genomic DNA was purified. Purified DNA was denatured with alkaline solution (0.1 N NaOH), spotted onto Hybond-N + membrane and blotted with anti-BrdU antibodies.

    Journal: Scientific Reports

    Article Title: The antibodies against 5-bromo-2′-deoxyuridine specifically recognize trifluridine incorporated into DNA

    doi: 10.1038/srep25286

    Figure Lengend Snippet: Detection of FTD by anti-BrdU antibodies. ( A ) Dot blot analysis of chemically synthesized FTD. BSA-conjugated FTD or non-conjugated (NC) BSA on nitrocellulose membrane were blotted with anti-BrdU antibodies. ( B ) Dot blot analysis of FTD or BrdU incorporated into the genomic DNA of HCT-116 cells. HCT-116 cells were cultured in the presence of 5 μM FTD or BrdU for 4 hours and genomic DNA was purified. Purified DNA was denatured with alkaline solution (0.1 N NaOH), spotted onto Hybond-N + membrane and blotted with anti-BrdU antibodies.

    Article Snippet: Equal amounts (100 ng, 10 ng, 1 ng, or 100 pg in 2 μL) of denatured (0.1 N NaOH for 5 minutes at room temperature (RT)) genomic DNA were spotted onto Hybond-N+ blotting membrane (GE Healthcare Life Sciences).

    Techniques: Dot Blot, Synthesized, Cell Culture, Purification

    Southern blot and PCR analyses of M. smegmatis LS2 strains. The genomic DNA was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to Hybond-N + membrane and hybridized by MSMEG_1556 probe. A. The expected DNA fragments hybridized by MSMEG_1556 probe were 2.12 kb and 2.55 kb. The expected size of PCR product from M. smegmatis LS2 strain was 3.15 kb. B. The expected DNA fragments hybridized by MSMEG_1556 probe was 3.37 kb and the expected size of PCR product from M. smegmatis mc 2 155 strain was 1.88 kb. C. Confirmation of M. smegmatis LS2 strains by Southern blot. Lanes 1–5. M. smegmatis LS2 strains have the hybridized DNA bands of 2.12 kb and 2.55 kb; lane 6. wild type M. smegmatis mc 2 155 has the hybridized DNA band of 3.37 kb. D. Confirmation of M. smegmatis LS2 strains by PCR. Lanes 1–5. the PCR product (3.15 kb) from M. smegmatis LS2 strains; lane 6. the PCR product (1.88 kb) from wild type M. smegmatis mc 2 155.

    Journal: PLoS ONE

    Article Title: Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

    doi: 10.1371/journal.pone.0042769

    Figure Lengend Snippet: Southern blot and PCR analyses of M. smegmatis LS2 strains. The genomic DNA was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to Hybond-N + membrane and hybridized by MSMEG_1556 probe. A. The expected DNA fragments hybridized by MSMEG_1556 probe were 2.12 kb and 2.55 kb. The expected size of PCR product from M. smegmatis LS2 strain was 3.15 kb. B. The expected DNA fragments hybridized by MSMEG_1556 probe was 3.37 kb and the expected size of PCR product from M. smegmatis mc 2 155 strain was 1.88 kb. C. Confirmation of M. smegmatis LS2 strains by Southern blot. Lanes 1–5. M. smegmatis LS2 strains have the hybridized DNA bands of 2.12 kb and 2.55 kb; lane 6. wild type M. smegmatis mc 2 155 has the hybridized DNA band of 3.37 kb. D. Confirmation of M. smegmatis LS2 strains by PCR. Lanes 1–5. the PCR product (3.15 kb) from M. smegmatis LS2 strains; lane 6. the PCR product (1.88 kb) from wild type M. smegmatis mc 2 155.

    Article Snippet: The DNA was then transferred to Hybond-N+ membrane (GE Healthcare).

    Techniques: Southern Blot, Polymerase Chain Reaction, Agarose Gel Electrophoresis