c fluminea tissues  (Qiagen)

 
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    Name:
    RNase Free DNase Set
    Description:
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    Catalog Number:
    79254
    Price:
    108
    Category:
    RNase Free DNase Set
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    Structured Review

    Qiagen c fluminea tissues
    RNase Free DNase Set
    For DNase digestion during RNA purification Kit contents Qiagen RNase free DNase Set 50 preps For DNase Digestion During RNA Purification Silica gel Membrane Spin column Technology Efficiently Removes the Majority of the DNA Without DNase Treatment The Buffer is Also Well suited for Efficient DNase Digestion in Solution Includes 1500U RNase free DNase I RNase free Buffer RDD and RNase free Water
    https://www.bioz.com/result/c fluminea tissues/product/Qiagen
    Average 85 stars, based on 38324 article reviews
    Price from $9.99 to $1999.99
    c fluminea tissues - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells"

    Article Title: Dynamics of Protein Phosphatase Gene Expression in Corbicula fluminea Exposed to Microcystin-LR and to Toxic Microcystis aeruginosa Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms12129172

    Projection of the normalized gene expression values of PPP1, PPP2 and PPP4 in C. fluminea visceral mass after exposure to 5 μg L −1 of MC-LR during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (PPP1: none; PPP2: Treatment; PPP4: none).
    Figure Legend Snippet: Projection of the normalized gene expression values of PPP1, PPP2 and PPP4 in C. fluminea visceral mass after exposure to 5 μg L −1 of MC-LR during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (PPP1: none; PPP2: Treatment; PPP4: none).

    Techniques Used: Expressing

    Tissue content (μg MC-LR g −1 DW) of unbound MC-LR in the visceral mass during exposure of C. fluminea to 5 μg L −1 MC-LR for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).
    Figure Legend Snippet: Tissue content (μg MC-LR g −1 DW) of unbound MC-LR in the visceral mass during exposure of C. fluminea to 5 μg L −1 MC-LR for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).

    Techniques Used:

    Projection of the normalized gene expression values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Exposure time squared , Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).
    Figure Legend Snippet: Projection of the normalized gene expression values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Exposure time squared , Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).

    Techniques Used: Expressing

    Tissue content (μg MC-LReq. g −1 DW) of unbound MC-LReq. in the visceral mass during exposure of C. fluminea to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).
    Figure Legend Snippet: Tissue content (μg MC-LReq. g −1 DW) of unbound MC-LReq. in the visceral mass during exposure of C. fluminea to 1 × 10 5 cells cm −3 of a M. aeruginosa toxic strain for 96 h. Values represent average of three replicates and bars represent confidence interval for mean level (95%).

    Techniques Used:

    Projection of enzyme activity values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells mL −1 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).
    Figure Legend Snippet: Projection of enzyme activity values of PPP2 in C. fluminea visceral mass after exposure to 1 × 10 5 cells mL −1 of a M. aeruginosa toxic strain during 96 h, in relation to the studied independent variables, according to the multiple linear regression analysis (Control groups-dashed line; Exposure groups-continuous line). The regression models describing the functions represented in the figure include only the significant regression variables (Treatment, Exposure time, Treatment × Exposure time, Treatment × Exposure time squared , Treatment × Exposure time cubed ).

    Techniques Used: Activity Assay

    2) Product Images from "Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses"

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01588

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.
    Figure Legend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Techniques Used: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    3) Product Images from "Investigation of G72 (DAOA) expression in the human brain"

    Article Title: Investigation of G72 (DAOA) expression in the human brain

    Journal: BMC Psychiatry

    doi: 10.1186/1471-244X-8-94

    Attempted detection of G72 protein in human samples by western blotting . Western blot membranes were probed with anti-G72 #1410 and #1411 as indicated above and visualised using the LI-COR Odyssey ® Infrared Imaging System. Lane 1: total protein lysate from His-G72-transfected HEK-293 cells (positive control). 2: no sample. 3–8: Clontech/BD Biosciences human
    Figure Legend Snippet: Attempted detection of G72 protein in human samples by western blotting . Western blot membranes were probed with anti-G72 #1410 and #1411 as indicated above and visualised using the LI-COR Odyssey ® Infrared Imaging System. Lane 1: total protein lysate from His-G72-transfected HEK-293 cells (positive control). 2: no sample. 3–8: Clontech/BD Biosciences human "protein medleys"; 3: spinal cord. 4: testis. 5: amygdala. 6: cerebellum. 7: frontal lobe. 8: fetal brain. 9: total protein lysate from rat cortex, postnatal day 14 (negative control – no G72 gene in rat). M: molecular weight standards. No specific signal at the expected size of ~18 kDa was detected in any of the human samples. A very weak signal at ~15 kDa was seen in testis, cerebellum and fetal brain (lanes 4, 6, 8). Anti-G72 #1411 detected a strong unspecific signal of unknown identity in all human CNS samples (lanes 3, 5, 6, 7, 8). Blots were reprobed with anti-actin as a loading control (lower panel).

    Techniques Used: Western Blot, Imaging, Transfection, Positive Control, Negative Control, Molecular Weight

    Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.
    Figure Legend Snippet: Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.

    Techniques Used: Expressing, Northern Blot, Isolation, Transfection

    Validation of G72 primer sets using real-time RT-PCR . (A) Primer sets G72 #1, #2, #3, #4 and #5, which were designed within the most common G72 exons (see table 1 and Fig. 2B ) were validated using human genomic DNA. Light grey: primer sequence within exon 4. Dark grey: primer sequence within exon 7. Hatched: primer sequence within exon 2. All primer sets gave a strong signal using human genomic DNA (A) and no signal using rat genomic DNA (not shown). (B) Primers sets within exon 2 (hatched) or exon 4 (light grey) readily detected a signal in cDNA that was isolated from HEK-293 cells expressing a His-tagged G72 cDNA, but not in mock-transfected cells (values within range of empty controls). The exon 7 reverse primer annealing sequence was not contained in our G72 cDNA construct and did thus not detect His-G72 (not shown).
    Figure Legend Snippet: Validation of G72 primer sets using real-time RT-PCR . (A) Primer sets G72 #1, #2, #3, #4 and #5, which were designed within the most common G72 exons (see table 1 and Fig. 2B ) were validated using human genomic DNA. Light grey: primer sequence within exon 4. Dark grey: primer sequence within exon 7. Hatched: primer sequence within exon 2. All primer sets gave a strong signal using human genomic DNA (A) and no signal using rat genomic DNA (not shown). (B) Primers sets within exon 2 (hatched) or exon 4 (light grey) readily detected a signal in cDNA that was isolated from HEK-293 cells expressing a His-tagged G72 cDNA, but not in mock-transfected cells (values within range of empty controls). The exon 7 reverse primer annealing sequence was not contained in our G72 cDNA construct and did thus not detect His-G72 (not shown).

    Techniques Used: Quantitative RT-PCR, Sequencing, Isolation, Expressing, Transfection, Construct

    Validation of anti-G72 antibodies using western blotting and LI-COR Odyssey ® Infrared Imaging System . (A) Western blotting was performed using rabbit polyclonal antibodies anti-G72 #1410 (left) or anti-G72 #1411 (right). Lane 1: total protein lysate from mock-transfected HEK-293 cells. Lane 2: total protein lysate from His-G72-transfected HEK-293 cells. Lane 3: Eluate from immunoprecipitation (IP) of mock lysate with anti-G72 #1411. Lane 4: Eluate from IP of His-G72 lysate with anti-G72 #1411. A strong signal for His-G72 was detected at ~22 kDa (lanes 2 and 4), with weaker bands around 18 kDa and below. Additional bands in lanes 3 and 4 (weak signal at 25 kDa and strong signal at 50 kDa) represent the light and heavy chain of the antibody used for IP. (B) G72-specific antibody #1410 (upper panel, green) and a commercial anti-His antibody (middle panel, red) detect the same protein bands on Western blots (merged image, lower panel, yellow) in total protein lysates from HEK-293 cells overexpressing His-G72. Lane 1 lane 2: as described in A.
    Figure Legend Snippet: Validation of anti-G72 antibodies using western blotting and LI-COR Odyssey ® Infrared Imaging System . (A) Western blotting was performed using rabbit polyclonal antibodies anti-G72 #1410 (left) or anti-G72 #1411 (right). Lane 1: total protein lysate from mock-transfected HEK-293 cells. Lane 2: total protein lysate from His-G72-transfected HEK-293 cells. Lane 3: Eluate from immunoprecipitation (IP) of mock lysate with anti-G72 #1411. Lane 4: Eluate from IP of His-G72 lysate with anti-G72 #1411. A strong signal for His-G72 was detected at ~22 kDa (lanes 2 and 4), with weaker bands around 18 kDa and below. Additional bands in lanes 3 and 4 (weak signal at 25 kDa and strong signal at 50 kDa) represent the light and heavy chain of the antibody used for IP. (B) G72-specific antibody #1410 (upper panel, green) and a commercial anti-His antibody (middle panel, red) detect the same protein bands on Western blots (merged image, lower panel, yellow) in total protein lysates from HEK-293 cells overexpressing His-G72. Lane 1 lane 2: as described in A.

    Techniques Used: Western Blot, Imaging, Transfection, Immunoprecipitation

    4) Product Images from "The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation"

    Article Title: The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0081-9

    Promoter activities of HML-10 LTRs. a LTRs of the HML-10(DAP3), HML-10(C4) and HML-10(PKIB) proviruses were cloned in both orientations into the promoter-free pGL3-Enhancer vector and transfected into HepG2 or HEK293T cells. Firefly luciferase (fLuc) activities were determined 24 h after transfection b Promoter activities expressed as fLuc activity normalized to renilla luciferase (rLuc) activity of the co-transfected pGL4.74 vector in the indicated cell lines. The pGL3-Control vector bearing the SV40 promoter ( grey bars ) served as positive and empty pGL3-Enhancer ( white bars ) as negative control. Promoter activities were normalized to pGL3-Control set to 100%. The bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test compared to pGL3-Enhancer. c For HepG2 cells the effect of IFNγ stimulation on two selected LTRs as well as the SV40 and HSV-TK promoters is shown. LTR and SV40 activity is expressed as fLuc normalized to rLuc signals, HSV-TK activity is expressed as rLuc activity only. The bars show mean ± SEM of at least three independent experiments and were normalized to unstimulated (-) cells set to 100%. n.d., not determined. d Identification of a conserved IFNγ activated site (GAS) of the consensus sequence 5′-TTNCNNNAA-3′ [ 45 ]. e Locations of primers used to detect transcripts originating from the 5′LTR of HML-10(DAP3). The predicted TSS was identified as described in the text and Additional file 1 : Figure S1. f Detection of DAP3 mRNA and HML-10(DAP3) transcripts in HepG2 and HeLa cells by qRT-PCR. cDNA samples prepared without reverse transcriptase (RT) for the indicated primer pairs, but with RT for GAPDH , served as controls. Values are normalized to GAPDH mRNA levels. Bars show mean ± SD of two measurements. In most cases, the SD is too small to be visible
    Figure Legend Snippet: Promoter activities of HML-10 LTRs. a LTRs of the HML-10(DAP3), HML-10(C4) and HML-10(PKIB) proviruses were cloned in both orientations into the promoter-free pGL3-Enhancer vector and transfected into HepG2 or HEK293T cells. Firefly luciferase (fLuc) activities were determined 24 h after transfection b Promoter activities expressed as fLuc activity normalized to renilla luciferase (rLuc) activity of the co-transfected pGL4.74 vector in the indicated cell lines. The pGL3-Control vector bearing the SV40 promoter ( grey bars ) served as positive and empty pGL3-Enhancer ( white bars ) as negative control. Promoter activities were normalized to pGL3-Control set to 100%. The bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test compared to pGL3-Enhancer. c For HepG2 cells the effect of IFNγ stimulation on two selected LTRs as well as the SV40 and HSV-TK promoters is shown. LTR and SV40 activity is expressed as fLuc normalized to rLuc signals, HSV-TK activity is expressed as rLuc activity only. The bars show mean ± SEM of at least three independent experiments and were normalized to unstimulated (-) cells set to 100%. n.d., not determined. d Identification of a conserved IFNγ activated site (GAS) of the consensus sequence 5′-TTNCNNNAA-3′ [ 45 ]. e Locations of primers used to detect transcripts originating from the 5′LTR of HML-10(DAP3). The predicted TSS was identified as described in the text and Additional file 1 : Figure S1. f Detection of DAP3 mRNA and HML-10(DAP3) transcripts in HepG2 and HeLa cells by qRT-PCR. cDNA samples prepared without reverse transcriptase (RT) for the indicated primer pairs, but with RT for GAPDH , served as controls. Values are normalized to GAPDH mRNA levels. Bars show mean ± SD of two measurements. In most cases, the SD is too small to be visible

    Techniques Used: Clone Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Negative Control, Sequencing, Quantitative RT-PCR

    Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit
    Figure Legend Snippet: Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit

    Techniques Used: Expressing, Sequencing, Allele-specific Oligonucleotide, Transfection, Quantitative RT-PCR, Light Microscopy

    5) Product Images from "The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation"

    Article Title: The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0081-9

    Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit
    Figure Legend Snippet: Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit

    Techniques Used: Expressing, Sequencing, Allele-specific Oligonucleotide, Transfection, Quantitative RT-PCR, Light Microscopy

    6) Product Images from "The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation"

    Article Title: The intron-enriched HERV-K(HML-10) family suppresses apoptosis, an indicator of malignant transformation

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0081-9

    Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit
    Figure Legend Snippet: Inactivating the HML-10(DAP3) RNA induces DAP3 expression and apoptosis in HeLa cells. a Target regions of sequence-specific ASOs are indicated. ASOs 1-4 are in antisense orientation to the retroviral transcript and in sense orientation to the DAP3 transcript. The ASO designated as Upstream served as control. b Cells were transfected with 25 or 50 nM of the indicated ASOs. At 24 h after transfection, expression levels of HML-10(DAP3) ( left ) and DAP3 mRNA ( right ) were determined by qRT-PCR. Bars show mean ± SEM of three independent experiments. RNA levels were normalized to GAPDH and levels of non-transfected cells were set to 1. * P -value ≤ 0.05, Student’s t -Test against Mock. c Cells were transfected with the indicated ASOs at 50 nM, after 24 h stimulated with 1000 U/mL IFNγ or 100 ng/mL TNFα, or left unstimulated. After additional 24 h, Trypan Blue exclusion as indicator of dead cells ( left ), MTS cell viability assays ( center ) or light microscopic analysis ( right ) was performed. Bars show mean ± SEM of three independent experiments in duplicates. * P -value ≤ 0.05, Student’s t -Test. The scale bar in light microscopy panel 1 is 100 μm. d Cells were transfected with the indicated ASOs at 50 nM. At 48 h after transfection, genomic DNA of these cells was prepared with the Apoptotic DNA Ladder Kit (Roche). The control DNA is from apoptotic U937 cells provided with the kit

    Techniques Used: Expressing, Sequencing, Allele-specific Oligonucleotide, Transfection, Quantitative RT-PCR, Light Microscopy

    7) Product Images from "Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation"

    Article Title: Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    Journal: Genome Research

    doi: 10.1101/gr.083931.108

    Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P
    Figure Legend Snippet: Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P

    Techniques Used: Expressing

    8) Product Images from "Investigation of G72 (DAOA) expression in the human brain"

    Article Title: Investigation of G72 (DAOA) expression in the human brain

    Journal: BMC Psychiatry

    doi: 10.1186/1471-244X-8-94

    Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.
    Figure Legend Snippet: Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.

    Techniques Used: Expressing, Northern Blot, Isolation, Transfection

    9) Product Images from "Epstein-Barr virus infection-induced inflammasome activation in human monocytes"

    Article Title: Epstein-Barr virus infection-induced inflammasome activation in human monocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175053

    EBV infection and inflammasome activation in human monocytes. (A) Confocal microscopic images of human monocytes 48 h after incubation with RPMI medium (no infection) or an AGS-EBV-GFP cell supernatant (AGSGFP: 6.0×10 6 or 6.0×10 5 GBU/mL). (B) EBV gene expression of human monocytes at 48 h after incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. EBV gene expression was quantified relative to beta-2-microglobulin expression. The data represent one experiment with triplicate samples. The error bars represent S.E. (C) IL-1β concentration in the supernatant of human monocytes over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. The data represent one experiment with duplicate samples. The error bars represent S.E. (D) Immunoblot analysis of caspase-1, IFI16, and AIM2 protein expression in human primary monocyte lysates over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. (E) Inflammasome-related gene (IL-1β, caspase-1, AIM2, NLRP3 and IFI16) expression of human monocytes over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant, was measured using RT-PCR. The data are expressed as fold changes compared to the 0-hour time-point. The data represent one experiment with triplicate samples. The error bars represent S.E. The asterisk (*) indicates p
    Figure Legend Snippet: EBV infection and inflammasome activation in human monocytes. (A) Confocal microscopic images of human monocytes 48 h after incubation with RPMI medium (no infection) or an AGS-EBV-GFP cell supernatant (AGSGFP: 6.0×10 6 or 6.0×10 5 GBU/mL). (B) EBV gene expression of human monocytes at 48 h after incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. EBV gene expression was quantified relative to beta-2-microglobulin expression. The data represent one experiment with triplicate samples. The error bars represent S.E. (C) IL-1β concentration in the supernatant of human monocytes over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. The data represent one experiment with duplicate samples. The error bars represent S.E. (D) Immunoblot analysis of caspase-1, IFI16, and AIM2 protein expression in human primary monocyte lysates over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant. (E) Inflammasome-related gene (IL-1β, caspase-1, AIM2, NLRP3 and IFI16) expression of human monocytes over time, following incubation with 6.0×10 6 GBU/mL of an AGSGFP cell supernatant, was measured using RT-PCR. The data are expressed as fold changes compared to the 0-hour time-point. The data represent one experiment with triplicate samples. The error bars represent S.E. The asterisk (*) indicates p

    Techniques Used: Infection, Activation Assay, Incubation, Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

    10) Product Images from "Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation"

    Article Title: Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    Journal: Genome Research

    doi: 10.1101/gr.083931.108

    Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P
    Figure Legend Snippet: Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P

    Techniques Used: Expressing

    11) Product Images from "Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation"

    Article Title: Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    Journal: Genome Research

    doi: 10.1101/gr.083931.108

    Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P
    Figure Legend Snippet: Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P

    Techniques Used: Expressing

    12) Product Images from "Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1"

    Article Title: Pseudoexfoliation syndrome-associated genetic variants affect transcription factor binding and alternative splicing of LOXL1

    Journal: Nature Communications

    doi: 10.1038/ncomms15466

    Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).
    Figure Legend Snippet: Effects of risk variants on LOXL1 transcriptional activity in vivo. ( a ) Scatter plot of TaqMan-based allelic discrimination of the LOXL1 SNP 13 (rs12441130). The genotypes of hTCF homoyzgous for the risk (C) or non-risk (T) alleles are shown in relation to genomic DNA and pre-mRNA containing cDNA of heterozygous hTCF cell lines ( n =15). Relative abundance of risk allele C over non-risk allele T in heterozygous hTCF cell lines ( n =15); expression level of T allele was set at 100%. ( b ) ChIP assay for RNA polymerase II (Pol II) binding at rs12441130 (SNP13)-containing region of LOXL1 in heterozygous hTCF cell lines ( n =2) using antibodies against Pol II, histone H3 and acetylated histone H3K27Ac (positive controls), and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. Allele-specific ChIP-qPCR analysis for Pol II chromatin binding and histone H3 is shown (left); expression levels of the non-risk allele T were set at 100%. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (top right) and by qPCR (bottom right) with primers specific for the SNP13 region producing a 121 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin).

    Techniques Used: Activity Assay, In Vivo, Expressing, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    13) Product Images from "Investigation of G72 (DAOA) expression in the human brain"

    Article Title: Investigation of G72 (DAOA) expression in the human brain

    Journal: BMC Psychiatry

    doi: 10.1186/1471-244X-8-94

    Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.
    Figure Legend Snippet: Investigation of G72 expression by northern analysis . (A) G72 and β-actin probes were validated using total RNA isolated from mock-transfected HEK-293 cells (M) or cells expressing His-G72 (G72). A strong signal for G72 was only observed in cells expressing His-G72. (B) Using a G72-specific, radioactive probe, no G72 signal was detected on Clontech poly A+ RNA Northern blots. The following brain regions were represented: Human Brain Blot II: 1, putamen. 2, temporal lobe. 3, frontal lobe. 4, occipital pole. 5, spinal cord. 6, medulla. 7, cerebral cortex. 8, cerebellum. Human Brain blot V: 9, thalamus. 10, whole brain. 11, hippocampus. 12, corpus callosum. 13, caudate nucleus. 14, amygdala. (C) Following G72-probing, human brain blots II and V were stripped and reprobed with a β-actin-specific probe in order to ensure RNA integrity and equal loading. Signal strength was similar in all lanes.

    Techniques Used: Expressing, Northern Blot, Isolation, Transfection

    14) Product Images from "β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum"

    Article Title: β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2016.07.162

    Promoter-based bioinformatic analysis of transcription factors in genes differentially-regulated in β-adrenergic-stimulated vs. control macrophages. Data represent mean fold difference (mean ratio of isoproterenol/control) ±SE of transcription
    Figure Legend Snippet: Promoter-based bioinformatic analysis of transcription factors in genes differentially-regulated in β-adrenergic-stimulated vs. control macrophages. Data represent mean fold difference (mean ratio of isoproterenol/control) ±SE of transcription

    Techniques Used:

    Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent
    Figure Legend Snippet: Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent

    Techniques Used: Activation Assay

    Mean diagnosticity score for genes that were up-regulated or down-regulated by β-adrenergic signaling. Diagnosticity scores for each gene transcript quantified the extent to which that transcript was predominately expressed by an M2-polarized
    Figure Legend Snippet: Mean diagnosticity score for genes that were up-regulated or down-regulated by β-adrenergic signaling. Diagnosticity scores for each gene transcript quantified the extent to which that transcript was predominately expressed by an M2-polarized

    Techniques Used:

    15) Product Images from "Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR"

    Article Title: Proteomic Analysis of the Quorum-Sensing Regulon in Pantoea stewartii and Identification of Direct Targets of EsaR

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01744-13

    DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I
    Figure Legend Snippet: DNase I footprinting assay of EsaR binding sites in the noncoding strand of P dkgA , coding strand of P glpF , and noncoding strand of P lrhA . (A to C) Capillary electrophoresis of FAM-labeled DNA fragments P dkgA (A), P glpF (B), and P lrhA (C) from DNase I

    Techniques Used: Footprinting, Binding Assay, Electrophoresis, Labeling

    Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding
    Figure Legend Snippet: Nested deletion EMSA analysis of EsaR direct targets. (A to C) The region protected by DNase I digestion in P dkgA (A), P glpF (B), and P lrhA (C) is the gray-shaded sequence (5′ to 3′), and the underlined bases are the 20-bp EsaR binding

    Techniques Used: Sequencing, Binding Assay

    16) Product Images from "LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells"

    Article Title: LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.200939677

    PreTCR and Notch signaling in LKB1-null thymocytes (A) Intracellular TCRβ expression in DN3 and DN4 thymocytes of LckCre + LKB1 +/+ and DN3 and CD44 − CD25 low thymocytes of LckCre + LKB1 fl/fl mice. (B) Flow cytometry of cellular DNA content (gated on live cells) of DN4 thymocyte from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice. The numbers indicate percentage of cells in S+G2/M phases of the cell cycle, representative of three independent experiments. (C) Data show CD98 and CD71 surface expression on DN4 thymocytes from LckCre + LKB1 +/+ and on CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (D) Histograms show Ser 235/236 S6 ribosomal protein phosphorylation in DN3 and DN4 thymocytes from LckCre + LKB1 +/+ and in DN3 and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (E) Quantitative RT-PCR examining the expression levels of CD2, CD5, Nur77, Hes1 and Deltex1 mRNA in DN4 thymocytes of LckCre + LKB1 +/+ ( n =3) and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl ( n =5) mice. Expression was normalized to the mRNA levels in LKB +/+ Lck-Cre + DN4 thymocytes; data show mean±SD. Differences in CD2 and CD5 expression are considered to be significant ( p =0.035 and p =0.036 for CD2 and CD5, respectively, p =0.1 for Nur77).
    Figure Legend Snippet: PreTCR and Notch signaling in LKB1-null thymocytes (A) Intracellular TCRβ expression in DN3 and DN4 thymocytes of LckCre + LKB1 +/+ and DN3 and CD44 − CD25 low thymocytes of LckCre + LKB1 fl/fl mice. (B) Flow cytometry of cellular DNA content (gated on live cells) of DN4 thymocyte from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice. The numbers indicate percentage of cells in S+G2/M phases of the cell cycle, representative of three independent experiments. (C) Data show CD98 and CD71 surface expression on DN4 thymocytes from LckCre + LKB1 +/+ and on CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (D) Histograms show Ser 235/236 S6 ribosomal protein phosphorylation in DN3 and DN4 thymocytes from LckCre + LKB1 +/+ and in DN3 and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (E) Quantitative RT-PCR examining the expression levels of CD2, CD5, Nur77, Hes1 and Deltex1 mRNA in DN4 thymocytes of LckCre + LKB1 +/+ ( n =3) and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl ( n =5) mice. Expression was normalized to the mRNA levels in LKB +/+ Lck-Cre + DN4 thymocytes; data show mean±SD. Differences in CD2 and CD5 expression are considered to be significant ( p =0.035 and p =0.036 for CD2 and CD5, respectively, p =0.1 for Nur77).

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR

    DN3/DN4 transition defects in LKB1-null thymocytes. (A) Flow cytometry of CD44 and CD25 surface expression in CD4 − CD8 − DN thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ thymi. CD25 and CD44 profiles of Thy-1 + DN cells additionally gated to exclude cells from the non-TCR lineage (lineage). Gates show DN1-DN4 populations, representative of five independent experiments. (B) Surface expression of CD25 in lineage Thy-1 + thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ mice, representative of five independent experiments. (C) Genomic PCR analysis of the LKB1 gene in DN3 and CD44 − CD25 low thymocytes. Top, PCR amplification of the gene product produced only when the LKB1 floxed allele is deleted in Lck-Cre + thymocytes. Bottom, PCR amplification of the non-deleted LKB1 gene. The upper bands represent the PCR product generated from the LKB1 non-deleted floxed gene, while the lower band indicates the WT LKB1 gene. (D) LckCre + LKB1 fl/fl CD44 − CD25 low thymocytes and LckCre + LKB1 +/+ DN4 thymocytes were either untreated or pretreated with STO-609 and then stimulated with 50 mM 2-deoxyglucose for 5 min. Western blot of cell lysates prepared from these thymocytes with pThr-172–AMPK and AMPK α1 antisera, representative of three independent experiments.
    Figure Legend Snippet: DN3/DN4 transition defects in LKB1-null thymocytes. (A) Flow cytometry of CD44 and CD25 surface expression in CD4 − CD8 − DN thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ thymi. CD25 and CD44 profiles of Thy-1 + DN cells additionally gated to exclude cells from the non-TCR lineage (lineage). Gates show DN1-DN4 populations, representative of five independent experiments. (B) Surface expression of CD25 in lineage Thy-1 + thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ mice, representative of five independent experiments. (C) Genomic PCR analysis of the LKB1 gene in DN3 and CD44 − CD25 low thymocytes. Top, PCR amplification of the gene product produced only when the LKB1 floxed allele is deleted in Lck-Cre + thymocytes. Bottom, PCR amplification of the non-deleted LKB1 gene. The upper bands represent the PCR product generated from the LKB1 non-deleted floxed gene, while the lower band indicates the WT LKB1 gene. (D) LckCre + LKB1 fl/fl CD44 − CD25 low thymocytes and LckCre + LKB1 +/+ DN4 thymocytes were either untreated or pretreated with STO-609 and then stimulated with 50 mM 2-deoxyglucose for 5 min. Western blot of cell lysates prepared from these thymocytes with pThr-172–AMPK and AMPK α1 antisera, representative of three independent experiments.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Polymerase Chain Reaction, Amplification, Produced, Generated, Western Blot

    LKB1 is required for survival of TCRβ selected T-cell progenitors A) DN thymocytes from LckCre + LKB1 +/+ ( n =3) and LckCre + LKB1 fl/fl ( n =3) mice were co-cultured with OP9-DL1 stromal cell monolayers. Cells were either stimulated with IL-7 or left untreated. Data show the mean fold increase in cell number following 5 days culture±SD. Differences in the proliferation of LckCre + LKB1 +/+ and LckCre + LKB1 fl/fl DN thymocytes are considered to be significant ( p =0.007 without IL-7 and p =0.0005 in the presence of IL-7, respectively). The IL-7-induced proliferation of LckCre + LKB1 fl/fl DN thymocytes is also significant, p =0.004. (B) DN thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers and treated with IL-7. The surface expression of CD4 and CD8 on thymocytes and the cell number were analyzed after 5 days of co-culture, representative of three independent experiments. C) DN4 thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1. Flow cytometric analysis of forward and side scatter after 18 and 60 h of co-culture, respectively. (D) DN4 thymocytes from tamoxifen LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 for 18 h, and stained with Thy1 and 7-aminoactinomycin D to investigate cell death. The numbers indicate the percentage of dead thymocytes. E) DN4 thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers. The data show the DNA content of DN4 thymocytes (not gated, total thymocyte population) after 18 h of co-culture. The numbers show the percentage of cells with degraded DNA.
    Figure Legend Snippet: LKB1 is required for survival of TCRβ selected T-cell progenitors A) DN thymocytes from LckCre + LKB1 +/+ ( n =3) and LckCre + LKB1 fl/fl ( n =3) mice were co-cultured with OP9-DL1 stromal cell monolayers. Cells were either stimulated with IL-7 or left untreated. Data show the mean fold increase in cell number following 5 days culture±SD. Differences in the proliferation of LckCre + LKB1 +/+ and LckCre + LKB1 fl/fl DN thymocytes are considered to be significant ( p =0.007 without IL-7 and p =0.0005 in the presence of IL-7, respectively). The IL-7-induced proliferation of LckCre + LKB1 fl/fl DN thymocytes is also significant, p =0.004. (B) DN thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers and treated with IL-7. The surface expression of CD4 and CD8 on thymocytes and the cell number were analyzed after 5 days of co-culture, representative of three independent experiments. C) DN4 thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1. Flow cytometric analysis of forward and side scatter after 18 and 60 h of co-culture, respectively. (D) DN4 thymocytes from tamoxifen LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 for 18 h, and stained with Thy1 and 7-aminoactinomycin D to investigate cell death. The numbers indicate the percentage of dead thymocytes. E) DN4 thymocytes from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice were co-cultured with OP9-DL1 stromal cell monolayers. The data show the DNA content of DN4 thymocytes (not gated, total thymocyte population) after 18 h of co-culture. The numbers show the percentage of cells with degraded DNA.

    Techniques Used: Mouse Assay, Cell Culture, Expressing, Co-Culture Assay, Flow Cytometry, Staining

    17) Product Images from "Knock-Down of CD44 Regulates Endothelial Cell Differentiation via NF?B-Mediated Chemokine Production"

    Article Title: Knock-Down of CD44 Regulates Endothelial Cell Differentiation via NF?B-Mediated Chemokine Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090921

    Characterisation of microvascular endothelial cells. (A) Phase contrast microscopy of TIME cells cultured on plastic dish or in Matrigel . TIME endothelial cells (2×10 5 ) were grown in 12-well plates under proliferating conditions on solid surface, or under differentiating conditions in Matrigel, for 16 h. (B) Expression of HAS, HYAL and CD44 under proliferating and differentiating conditions . Expression levels relative to GAPDH of HAS1, 2, 3, HYAL1, 2 and CD44s, v3, v6 mRNAs were determined by real time PCR, as described in Materials and Methods . Results are mean of three separate experiments performed in triplicates ± S.D. (C) Detection of knock-down of HYAL2 or CD44 at mRNA and protein level . Endothelial cells were transfected with 5 nM of siRNA targeting either HYAL2 or CD44. RNA was extracted after 24 h and subjected to real time PCR to determine the knock-down efficiency at the mRNA level. For Western blotting, cells were lysed after 48 h of transfection and subjected to SDS-PAGE (insert). (D) Hyaluronan production and hyaluronidase activity . TIME cells were transfected with siRNAs (scrambled control, HYAL2 or CD44) for 24 h. Then, medium was changed to growth medium containing or not containing 800 ng hyaluronan/1 million cells. Conditioned media were collected after 24 h and the hyaluronan content detected, as described in Materials and Methods . A representative experiment, out of three separate experiments performed with similar results is shown.
    Figure Legend Snippet: Characterisation of microvascular endothelial cells. (A) Phase contrast microscopy of TIME cells cultured on plastic dish or in Matrigel . TIME endothelial cells (2×10 5 ) were grown in 12-well plates under proliferating conditions on solid surface, or under differentiating conditions in Matrigel, for 16 h. (B) Expression of HAS, HYAL and CD44 under proliferating and differentiating conditions . Expression levels relative to GAPDH of HAS1, 2, 3, HYAL1, 2 and CD44s, v3, v6 mRNAs were determined by real time PCR, as described in Materials and Methods . Results are mean of three separate experiments performed in triplicates ± S.D. (C) Detection of knock-down of HYAL2 or CD44 at mRNA and protein level . Endothelial cells were transfected with 5 nM of siRNA targeting either HYAL2 or CD44. RNA was extracted after 24 h and subjected to real time PCR to determine the knock-down efficiency at the mRNA level. For Western blotting, cells were lysed after 48 h of transfection and subjected to SDS-PAGE (insert). (D) Hyaluronan production and hyaluronidase activity . TIME cells were transfected with siRNAs (scrambled control, HYAL2 or CD44) for 24 h. Then, medium was changed to growth medium containing or not containing 800 ng hyaluronan/1 million cells. Conditioned media were collected after 24 h and the hyaluronan content detected, as described in Materials and Methods . A representative experiment, out of three separate experiments performed with similar results is shown.

    Techniques Used: Microscopy, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot, SDS Page, Activity Assay

    Gene expression by TIME cells undergoing tubulogenesis upon knockdown of HYAL2 or CD44. Microvascular endothelial cells transfected with siRNA for scrambled control, HYAL2 or CD44 were grown under differentiating conditions in Matrigel for 16-change lower than 0.5 were considered as downregulated, whereas genes with a fold-change above 2 were considered as upregulated.
    Figure Legend Snippet: Gene expression by TIME cells undergoing tubulogenesis upon knockdown of HYAL2 or CD44. Microvascular endothelial cells transfected with siRNA for scrambled control, HYAL2 or CD44 were grown under differentiating conditions in Matrigel for 16-change lower than 0.5 were considered as downregulated, whereas genes with a fold-change above 2 were considered as upregulated.

    Techniques Used: Expressing, Transfection

    Knockdown of HYAL2 or CD44 impairs tubulogenesis. TIME cells were transfected with scrambled control siRNA or siRNAs for HYAL2 or CD44 for 24 h, before seeded onto Matrigel. Phase contrast overview photos, using a Zeiss Axiovert40 microscope, were taken at the indicated time points. A representative experiment out of three experiments performed in duplicates is depicted. Scale bar, 200 µm.
    Figure Legend Snippet: Knockdown of HYAL2 or CD44 impairs tubulogenesis. TIME cells were transfected with scrambled control siRNA or siRNAs for HYAL2 or CD44 for 24 h, before seeded onto Matrigel. Phase contrast overview photos, using a Zeiss Axiovert40 microscope, were taken at the indicated time points. A representative experiment out of three experiments performed in duplicates is depicted. Scale bar, 200 µm.

    Techniques Used: Transfection, Microscopy

    Gene expression levels of CXCL9 and CXCL12 and their receptors upon knockdown of CD44 or HYAL2 under proliferating and differentiating conditions. TIME cells transfected with scrambled control siRNA or siRNA against CD44 or HYAL2 were grown on Matrigel or on plastic dishes. RNA was extracted, reversely transcribed and subjected to real time PCR. Gene expression of the chemokines CXCL9 and CXCL12 and their receptors CXCR3 and CXCR4, respectively, were determined, as described in Materials and Methods . A representative experiment out of three performed in triplicates with similar results is shown ± SD.
    Figure Legend Snippet: Gene expression levels of CXCL9 and CXCL12 and their receptors upon knockdown of CD44 or HYAL2 under proliferating and differentiating conditions. TIME cells transfected with scrambled control siRNA or siRNA against CD44 or HYAL2 were grown on Matrigel or on plastic dishes. RNA was extracted, reversely transcribed and subjected to real time PCR. Gene expression of the chemokines CXCL9 and CXCL12 and their receptors CXCR3 and CXCR4, respectively, were determined, as described in Materials and Methods . A representative experiment out of three performed in triplicates with similar results is shown ± SD.

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction

    18) Product Images from "β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum"

    Article Title: β-adrenergic-stimulated macrophages: Comprehensive localization in the M1–M2 spectrum

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2016.07.162

    Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent
    Figure Legend Snippet: Effect of β-adrenergic signaling on select gene transcripts that constitute macrophage activation categories along the M1–M2 spectrum in Murray et al., 2014. Isoproterenol at 1 μM. Data represent mean ± SE of three independent

    Techniques Used: Activation Assay

    (A) M1 - M2 spectrum categories of macrophage activation, based on the activator and subsequent known transcription factors, as proposed by Peter J. Murray and colleagues following the International Congress of Immunology in Milan in 2013. At the far
    Figure Legend Snippet: (A) M1 - M2 spectrum categories of macrophage activation, based on the activator and subsequent known transcription factors, as proposed by Peter J. Murray and colleagues following the International Congress of Immunology in Milan in 2013. At the far

    Techniques Used: Activation Assay

    19) Product Images from "LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells"

    Article Title: LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells

    Journal: European Journal of Immunology

    doi: 10.1002/eji.200939677

    PreTCR and Notch signaling in LKB1-null thymocytes (A) Intracellular TCRβ expression in DN3 and DN4 thymocytes of LckCre + LKB1 +/+ and DN3 and CD44 − CD25 low thymocytes of LckCre + LKB1 fl/fl mice. (B) Flow cytometry of cellular DNA content (gated on live cells) of DN4 thymocyte from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice. The numbers indicate percentage of cells in S+G2/M phases of the cell cycle, representative of three independent experiments. (C) Data show CD98 and CD71 surface expression on DN4 thymocytes from LckCre + LKB1 +/+ and on CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (D) Histograms show Ser 235/236 S6 ribosomal protein phosphorylation in DN3 and DN4 thymocytes from LckCre + LKB1 +/+ and in DN3 and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (E) Quantitative RT-PCR examining the expression levels of CD2, CD5, Nur77, Hes1 and Deltex1 mRNA in DN4 thymocytes of LckCre + LKB1 +/+ ( n =3) and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl ( n =5) mice. Expression was normalized to the mRNA levels in LKB +/+ Lck-Cre + DN4 thymocytes; data show mean±SD. Differences in CD2 and CD5 expression are considered to be significant ( p =0.035 and p =0.036 for CD2 and CD5, respectively, p =0.1 for Nur77).
    Figure Legend Snippet: PreTCR and Notch signaling in LKB1-null thymocytes (A) Intracellular TCRβ expression in DN3 and DN4 thymocytes of LckCre + LKB1 +/+ and DN3 and CD44 − CD25 low thymocytes of LckCre + LKB1 fl/fl mice. (B) Flow cytometry of cellular DNA content (gated on live cells) of DN4 thymocyte from LckCre + LKB1 +/+ and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice. The numbers indicate percentage of cells in S+G2/M phases of the cell cycle, representative of three independent experiments. (C) Data show CD98 and CD71 surface expression on DN4 thymocytes from LckCre + LKB1 +/+ and on CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (D) Histograms show Ser 235/236 S6 ribosomal protein phosphorylation in DN3 and DN4 thymocytes from LckCre + LKB1 +/+ and in DN3 and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl mice, representative of three independent experiments. (E) Quantitative RT-PCR examining the expression levels of CD2, CD5, Nur77, Hes1 and Deltex1 mRNA in DN4 thymocytes of LckCre + LKB1 +/+ ( n =3) and CD44 − CD25 low thymocytes from LckCre + LKB1 fl/fl ( n =5) mice. Expression was normalized to the mRNA levels in LKB +/+ Lck-Cre + DN4 thymocytes; data show mean±SD. Differences in CD2 and CD5 expression are considered to be significant ( p =0.035 and p =0.036 for CD2 and CD5, respectively, p =0.1 for Nur77).

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR

    DN3/DN4 transition defects in LKB1-null thymocytes. (A) Flow cytometry of CD44 and CD25 surface expression in CD4 − CD8 − DN thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ thymi. CD25 and CD44 profiles of Thy-1 + DN cells additionally gated to exclude cells from the non-TCR lineage (lineage). Gates show DN1-DN4 populations, representative of five independent experiments. (B) Surface expression of CD25 in lineage Thy-1 + thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ mice, representative of five independent experiments. (C) Genomic PCR analysis of the LKB1 gene in DN3 and CD44 − CD25 low thymocytes. Top, PCR amplification of the gene product produced only when the LKB1 floxed allele is deleted in Lck-Cre + thymocytes. Bottom, PCR amplification of the non-deleted LKB1 gene. The upper bands represent the PCR product generated from the LKB1 non-deleted floxed gene, while the lower band indicates the WT LKB1 gene. (D) LckCre + LKB1 fl/fl CD44 − CD25 low thymocytes and LckCre + LKB1 +/+ DN4 thymocytes were either untreated or pretreated with STO-609 and then stimulated with 50 mM 2-deoxyglucose for 5 min. Western blot of cell lysates prepared from these thymocytes with pThr-172–AMPK and AMPK α1 antisera, representative of three independent experiments.
    Figure Legend Snippet: DN3/DN4 transition defects in LKB1-null thymocytes. (A) Flow cytometry of CD44 and CD25 surface expression in CD4 − CD8 − DN thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ thymi. CD25 and CD44 profiles of Thy-1 + DN cells additionally gated to exclude cells from the non-TCR lineage (lineage). Gates show DN1-DN4 populations, representative of five independent experiments. (B) Surface expression of CD25 in lineage Thy-1 + thymocytes from LckCre + LKB1 fl/fl and LckCre + LKB1 +/+ mice, representative of five independent experiments. (C) Genomic PCR analysis of the LKB1 gene in DN3 and CD44 − CD25 low thymocytes. Top, PCR amplification of the gene product produced only when the LKB1 floxed allele is deleted in Lck-Cre + thymocytes. Bottom, PCR amplification of the non-deleted LKB1 gene. The upper bands represent the PCR product generated from the LKB1 non-deleted floxed gene, while the lower band indicates the WT LKB1 gene. (D) LckCre + LKB1 fl/fl CD44 − CD25 low thymocytes and LckCre + LKB1 +/+ DN4 thymocytes were either untreated or pretreated with STO-609 and then stimulated with 50 mM 2-deoxyglucose for 5 min. Western blot of cell lysates prepared from these thymocytes with pThr-172–AMPK and AMPK α1 antisera, representative of three independent experiments.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Polymerase Chain Reaction, Amplification, Produced, Generated, Western Blot

    20) Product Images from "Heme Uptake Mediated by LHR1 Is Essential for Leishmania amazonensis Virulence"

    Article Title: Heme Uptake Mediated by LHR1 Is Essential for Leishmania amazonensis Virulence

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00687-13

    LHR1 deficiency does not affect the generation of axenic amastigotes. Amastigotes of L. amazonensis were obtained axenically (see Materials and Methods) and cultivated for 5 days. (A) The percentage of round aflagellated forms was determined microscopically,
    Figure Legend Snippet: LHR1 deficiency does not affect the generation of axenic amastigotes. Amastigotes of L. amazonensis were obtained axenically (see Materials and Methods) and cultivated for 5 days. (A) The percentage of round aflagellated forms was determined microscopically,

    Techniques Used:

    LHR1/ Δ lhr1 amastigotes are unable to proliferate in BMM. (A) BMM left untreated (Control) or preincubated with red blood cells (RBC) were infected with L. amazonensis axenic amastigotes (multiplicity of infection [MOI] = 5) for 1, 24, 48, and
    Figure Legend Snippet: LHR1/ Δ lhr1 amastigotes are unable to proliferate in BMM. (A) BMM left untreated (Control) or preincubated with red blood cells (RBC) were infected with L. amazonensis axenic amastigotes (multiplicity of infection [MOI] = 5) for 1, 24, 48, and

    Techniques Used: Infection

    21) Product Images from "A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont"

    Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

    Journal: Cell

    doi: 10.1016/j.cell.2009.02.041

    Subcellular Localization of the Glycoproteins (A) Cytoplasmic and periplasmic fractions of wild-type B. fragilis , separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli . (B) Cytoplasmic and periplasmic fractions from B. fragilis expressing His-tagged BF2494, BF0447, BF0935 or BF2334, separated by SDS-PAGE, blotted and probed with anti-His-tag. (C) Intact B. fragilis Δ tsr15 M8 cells were incubated with proteinase K for the indicated times and the lysate separated by SDS-PAGE, blotted and probed with antisera to His-tagged BF3567, AapA, or BF2494 purified from E. coli . BF2494 and AapA are controls located in the periplasm or on the surface of the cell, respectively. (D) Wild-type B. fragilis expressing BF0522-His or BF3918-His were incubated with proteinase K for the indicated times, lysed, separated by SDS-PAGE, blotted and probed with anti-His-tag.
    Figure Legend Snippet: Subcellular Localization of the Glycoproteins (A) Cytoplasmic and periplasmic fractions of wild-type B. fragilis , separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli . (B) Cytoplasmic and periplasmic fractions from B. fragilis expressing His-tagged BF2494, BF0447, BF0935 or BF2334, separated by SDS-PAGE, blotted and probed with anti-His-tag. (C) Intact B. fragilis Δ tsr15 M8 cells were incubated with proteinase K for the indicated times and the lysate separated by SDS-PAGE, blotted and probed with antisera to His-tagged BF3567, AapA, or BF2494 purified from E. coli . BF2494 and AapA are controls located in the periplasm or on the surface of the cell, respectively. (D) Wild-type B. fragilis expressing BF0522-His or BF3918-His were incubated with proteinase K for the indicated times, lysed, separated by SDS-PAGE, blotted and probed with anti-His-tag.

    Techniques Used: SDS Page, Purification, Expressing, Incubation

    Site-Directed Mutagenesis of BF2494 (A) Amino acid sequence of BF2494. The signal peptide is underlined. Segments in upper case and bolded are S/T-containing tryptic peptides that were not detected in at least 2 of 6 MS analyses of purified BF2494-His (excluding the signal peptide). Boxed or underlined residues in these segments were mutated to alanine to determine whether they are glycosylated; boxed residues were found to be glycosylated and underlined residues were not. (B) Wild-type (WT) and mutant BF2494-His proteins purified from B. fragilis , separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli or stained with Coomassie Blue. (C) Same as panel B except that the gel was either stained with Pro-Q Emerald Glycostain, or blotted and probed with AAL or the antiserum to the glycan of BF2494. (D) Cytoplasmic and periplasmic fractions of B. fragilis ). (E) Same as panel D except that the protein is the triple T→A BF2494-His molecule. (F) Alignment of protein sequences surrounding the three glycosylation sites of BF2494. Glycosylated residues are underlined and conserved or similar residues are bold. (G) Whole cell lysates of B. fragilis expressing wild-type and various mutant BF2494-His proteins, separated by SDS-PAGE, blotted and probed with antibody to the His-tag.
    Figure Legend Snippet: Site-Directed Mutagenesis of BF2494 (A) Amino acid sequence of BF2494. The signal peptide is underlined. Segments in upper case and bolded are S/T-containing tryptic peptides that were not detected in at least 2 of 6 MS analyses of purified BF2494-His (excluding the signal peptide). Boxed or underlined residues in these segments were mutated to alanine to determine whether they are glycosylated; boxed residues were found to be glycosylated and underlined residues were not. (B) Wild-type (WT) and mutant BF2494-His proteins purified from B. fragilis , separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli or stained with Coomassie Blue. (C) Same as panel B except that the gel was either stained with Pro-Q Emerald Glycostain, or blotted and probed with AAL or the antiserum to the glycan of BF2494. (D) Cytoplasmic and periplasmic fractions of B. fragilis ). (E) Same as panel D except that the protein is the triple T→A BF2494-His molecule. (F) Alignment of protein sequences surrounding the three glycosylation sites of BF2494. Glycosylated residues are underlined and conserved or similar residues are bold. (G) Whole cell lysates of B. fragilis expressing wild-type and various mutant BF2494-His proteins, separated by SDS-PAGE, blotted and probed with antibody to the His-tag.

    Techniques Used: Mutagenesis, Sequencing, Mass Spectrometry, Purification, SDS Page, Staining, Expressing

    A Genomic Region Involved in Protein Glycosylation (A) The region of the B. fragilis genome containing metG and genes BF4298-4306. Putative glycosyltransferase genes are hatched. (B) Phosphoimager scan of SDS-PAGE-separated whole cell lysates of wild-type, Δ gmd-fcl Δ fkp , and Δ(BF4298-4306) grown in a medium containing 3 H-fucose. (C) Whole cell lysates of wild-type and mutants separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli . (D) Whole cell lysates of wild-type and mutant B. fragilis expressing BF0447-His or BF0522-His, separated by SDS-PAGE, blotted and probed with anti-His-tag. (E) RT-PCR amplification of a 1142 bp region including portions of the coding regions of metG and wzx, as indicated in panel A. Reactions were performed with and without reverse transcriptase (RT).
    Figure Legend Snippet: A Genomic Region Involved in Protein Glycosylation (A) The region of the B. fragilis genome containing metG and genes BF4298-4306. Putative glycosyltransferase genes are hatched. (B) Phosphoimager scan of SDS-PAGE-separated whole cell lysates of wild-type, Δ gmd-fcl Δ fkp , and Δ(BF4298-4306) grown in a medium containing 3 H-fucose. (C) Whole cell lysates of wild-type and mutants separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli . (D) Whole cell lysates of wild-type and mutant B. fragilis expressing BF0447-His or BF0522-His, separated by SDS-PAGE, blotted and probed with anti-His-tag. (E) RT-PCR amplification of a 1142 bp region including portions of the coding regions of metG and wzx, as indicated in panel A. Reactions were performed with and without reverse transcriptase (RT).

    Techniques Used: SDS Page, Purification, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

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    Isolation:

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    Purification:

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