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Bio-Rad version 4 4 0 software
Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.
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1) Product Images from "Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly"

Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-8-9

Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.
Figure Legend Snippet: Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Incubation, Isolation, Western Blot, Software

2) Product Images from "PiggyBac Transposable Element-derived 1 controls Neuronal Progenitor Identity, Stress Sensing and mammal-specific paraspeckles"

Article Title: PiggyBac Transposable Element-derived 1 controls Neuronal Progenitor Identity, Stress Sensing and mammal-specific paraspeckles

Journal: bioRxiv

doi: 10.1101/2021.05.19.444448

The domesticated PGBD1 possesses a SCAN-, KRAB- and transposase-derived domains, but has no catalytic activity as a transposase a, PGBD1 domain structure in comparison to PiggyBac of the cabbage looper, human PGBD2, rat PGBD1 and mouse PGBD1. The transposase-derived domain (IS4) includes dimerization and DNA binding domains (DDBD) as well as the catalytic domains of PiggyBac 80 . NTD, N-terminal domain; CRD, C-terminal cysteine rich domain; E1-7 are exons 1 to 7. The ‘D’s in the transposase-derived domains represent the catalytic triad DDD (D268, D346, D447). D447 is replaced by A in PGBD1. PGBD2 and PGBD1 are highly similar (average pairwise similarity score of ∼ 63% the aligned region which spans 1324 bp exceeds the borders of the annotated transposase IS4 domain, calculated by distance matrix of Ugene). Note that the ZN-finger containing CRD domain, required for ITR binding in the piggyBac transposase is missing in PGBD1 223 . The PGBD1 sequences in rodent animal models are truncated, resulting in degenerated copies. The Ka/Kv values of the entire PGBD1 as well as for various subdomains are shown. Note the ∼1 value for the KRAB domain. (overall = 0.35, N-terminal (aa 1-290) = 0.56, C-terminal (aa 291-809) = 0.21, SCAN (aa 40-142) = 0.32, KRAB (aa 211-267) = 1.02, DDBD1 (aa 405-541) = 0.19, DDBD2 (aa 750-804) = 0.26, catalytic domain 1 (aa 541-651) = 0.14 and catalytic domain 2 (aa 726-750) = 0.07, reference is the human amino acid sequence of PGBD1). b, Phylogenetic tree of PGBD1 and PGBD2. The presence of the transposase-derived, the SCAN and KRAB domains are shown. The human PGBD1 and PGBD2, with the most closely related sequences (containing transposase IS4) were aligned with muscle and a tree was built using MrBayes . Protein domains were annotated with hmmerscan and CDD (NCBI). The KRAB domain was annotated with Phyre2. c, Relative expression levels of PGBD1 and PGBD2 in human and chimpanzee NPCs and neurons 73 (GSE83638). Note that the in cross-species comparison the expression of PGBD1 and PGBD2 is specifically enriched in human and chimp, respectively. d, Protein sequence alignment of the transposase-derived DDD catalytic domain of PGBD1. The first raw of the alignment shows the corresponding sequence of the piggyBac transposase, identified in Trichoplusiani (cabbage looper). The alignment includes koala and grey seal, from where the KRAB domain was reported (see also Fig. S1B), and various mammalian species. The conserved amino acids D268/D346/D447 of the conserved DDD catalytic domain and D450 of the piggyBac transposase are arrowed 46 . The numbers refer to their position using the piggyBac amino acid sequence as reference. e, Transposon excision repair assay detects no activity of the PGBD1. (Left panel) Schematic representation of the reporter assay of PiggyBac excision. The PiggyBac transposon (flanked by inverted terminal repeats, ITRs) splits the coding sequence of the green fluorescence protein (GFP) reporter. In the presence of an active transposase, transposon excision occurs, and the readout is the restored GFP reporter signal. (Middle panel) Quantitative FACS analysis of GFP positive cells generated in the transposon excision repair assay. HeLa cells were co-transfected with plasmids harbouring HA-tagged PGBD1 along with the reporter construct. Non-transfected HeLa and cells transfected with mPB (codon optimized, mouse piggyBac transposase) along with the reporter served as controls. (Right panel) Western blot analysis of the HA-tagged PGBD1 (HA-PGBD1) protein tested in the excision repair assay. f, Transposition assay detects no activity of the PGBD1 protein. (Left panel) Schematic representation of the colony forming transposition assay to detect stable integration of the puromycin resistance gene marked reporter in HEK293 cells. In case of active transposition, the transposase cuts at the ITRs - (inverted terminal repeats), and inserts the reporter-marked transposon into the genome, providing antibiotic resistance for the transfected cells. In addition to the piggyBac ITRs, reporters were also built with PiggyBac-derived miniature inverted repeat (MITEs) ITRs of MER75, MER85. See also Fig. 2a . (Right panel) Puromycin resistant HEK293 colonies as the readout of the assay. The constructs were transfected in various combinations. HEK293 cells transfected with the mPB transposase and the non-relevant Luciferase expression construct (Luc) along with the reporter served as controls. (Bottom Left panel) Quantification of the transposition assay. Colonies were quantified in a 75S model gel imager, using the Quantity One 4.4.0 software (Bio-Rad). Error bars indicate s.d . Note that the mPB transposase (positive control) was able to mobilize the PB transposon, but not the PB-inverted repeat related reporters.
Figure Legend Snippet: The domesticated PGBD1 possesses a SCAN-, KRAB- and transposase-derived domains, but has no catalytic activity as a transposase a, PGBD1 domain structure in comparison to PiggyBac of the cabbage looper, human PGBD2, rat PGBD1 and mouse PGBD1. The transposase-derived domain (IS4) includes dimerization and DNA binding domains (DDBD) as well as the catalytic domains of PiggyBac 80 . NTD, N-terminal domain; CRD, C-terminal cysteine rich domain; E1-7 are exons 1 to 7. The ‘D’s in the transposase-derived domains represent the catalytic triad DDD (D268, D346, D447). D447 is replaced by A in PGBD1. PGBD2 and PGBD1 are highly similar (average pairwise similarity score of ∼ 63% the aligned region which spans 1324 bp exceeds the borders of the annotated transposase IS4 domain, calculated by distance matrix of Ugene). Note that the ZN-finger containing CRD domain, required for ITR binding in the piggyBac transposase is missing in PGBD1 223 . The PGBD1 sequences in rodent animal models are truncated, resulting in degenerated copies. The Ka/Kv values of the entire PGBD1 as well as for various subdomains are shown. Note the ∼1 value for the KRAB domain. (overall = 0.35, N-terminal (aa 1-290) = 0.56, C-terminal (aa 291-809) = 0.21, SCAN (aa 40-142) = 0.32, KRAB (aa 211-267) = 1.02, DDBD1 (aa 405-541) = 0.19, DDBD2 (aa 750-804) = 0.26, catalytic domain 1 (aa 541-651) = 0.14 and catalytic domain 2 (aa 726-750) = 0.07, reference is the human amino acid sequence of PGBD1). b, Phylogenetic tree of PGBD1 and PGBD2. The presence of the transposase-derived, the SCAN and KRAB domains are shown. The human PGBD1 and PGBD2, with the most closely related sequences (containing transposase IS4) were aligned with muscle and a tree was built using MrBayes . Protein domains were annotated with hmmerscan and CDD (NCBI). The KRAB domain was annotated with Phyre2. c, Relative expression levels of PGBD1 and PGBD2 in human and chimpanzee NPCs and neurons 73 (GSE83638). Note that the in cross-species comparison the expression of PGBD1 and PGBD2 is specifically enriched in human and chimp, respectively. d, Protein sequence alignment of the transposase-derived DDD catalytic domain of PGBD1. The first raw of the alignment shows the corresponding sequence of the piggyBac transposase, identified in Trichoplusiani (cabbage looper). The alignment includes koala and grey seal, from where the KRAB domain was reported (see also Fig. S1B), and various mammalian species. The conserved amino acids D268/D346/D447 of the conserved DDD catalytic domain and D450 of the piggyBac transposase are arrowed 46 . The numbers refer to their position using the piggyBac amino acid sequence as reference. e, Transposon excision repair assay detects no activity of the PGBD1. (Left panel) Schematic representation of the reporter assay of PiggyBac excision. The PiggyBac transposon (flanked by inverted terminal repeats, ITRs) splits the coding sequence of the green fluorescence protein (GFP) reporter. In the presence of an active transposase, transposon excision occurs, and the readout is the restored GFP reporter signal. (Middle panel) Quantitative FACS analysis of GFP positive cells generated in the transposon excision repair assay. HeLa cells were co-transfected with plasmids harbouring HA-tagged PGBD1 along with the reporter construct. Non-transfected HeLa and cells transfected with mPB (codon optimized, mouse piggyBac transposase) along with the reporter served as controls. (Right panel) Western blot analysis of the HA-tagged PGBD1 (HA-PGBD1) protein tested in the excision repair assay. f, Transposition assay detects no activity of the PGBD1 protein. (Left panel) Schematic representation of the colony forming transposition assay to detect stable integration of the puromycin resistance gene marked reporter in HEK293 cells. In case of active transposition, the transposase cuts at the ITRs - (inverted terminal repeats), and inserts the reporter-marked transposon into the genome, providing antibiotic resistance for the transfected cells. In addition to the piggyBac ITRs, reporters were also built with PiggyBac-derived miniature inverted repeat (MITEs) ITRs of MER75, MER85. See also Fig. 2a . (Right panel) Puromycin resistant HEK293 colonies as the readout of the assay. The constructs were transfected in various combinations. HEK293 cells transfected with the mPB transposase and the non-relevant Luciferase expression construct (Luc) along with the reporter served as controls. (Bottom Left panel) Quantification of the transposition assay. Colonies were quantified in a 75S model gel imager, using the Quantity One 4.4.0 software (Bio-Rad). Error bars indicate s.d . Note that the mPB transposase (positive control) was able to mobilize the PB transposon, but not the PB-inverted repeat related reporters.

Techniques Used: Derivative Assay, Activity Assay, Binding Assay, Sequencing, Expressing, Reporter Assay, Fluorescence, FACS, Generated, Transfection, Construct, Western Blot, Luciferase, Software, Positive Control

3) Product Images from "Changes in expression of VE-cadherin and MMPs in endothelial cells: Implications for angiogenesis"

Article Title: Changes in expression of VE-cadherin and MMPs in endothelial cells: Implications for angiogenesis

Journal: Vascular Cell

doi: 10.1186/2045-824X-3-6

Effect of modulators of angiogenesis on the activity and production of MMPs : Equivalent aliquots of the medium normalized to the total cell protein from the culture of control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells on days 1 to 4 were subjected to zymography (A) as described in the text.( a ) Lanes 1-4, 50 μM ursolic acid treated cells 1 st -4 th day, Lanes 5-8, control 1 st -4 th day. ( b ) Lanes 1-4, 10 μM curcumin treated cells 1 st -4 th day, Lanes 5-8, aspirin treated cells 1 st -4 th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm 2 /mg protein (B) . Values given are the average of intensity measurement of 6 experiments ± SEM. The culture medium collected from control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells at intervals of 24 hrs (normalized to the total cell protein) were quantitated for MMP-9 and MMP-2 protein expression by ELISA using specific antibody ( C ) . Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day (p
Figure Legend Snippet: Effect of modulators of angiogenesis on the activity and production of MMPs : Equivalent aliquots of the medium normalized to the total cell protein from the culture of control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells on days 1 to 4 were subjected to zymography (A) as described in the text.( a ) Lanes 1-4, 50 μM ursolic acid treated cells 1 st -4 th day, Lanes 5-8, control 1 st -4 th day. ( b ) Lanes 1-4, 10 μM curcumin treated cells 1 st -4 th day, Lanes 5-8, aspirin treated cells 1 st -4 th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm 2 /mg protein (B) . Values given are the average of intensity measurement of 6 experiments ± SEM. The culture medium collected from control, ursolic acid (50 μM), curcumin (10 μM) and aspirin (100 μM) treated cells at intervals of 24 hrs (normalized to the total cell protein) were quantitated for MMP-9 and MMP-2 protein expression by ELISA using specific antibody ( C ) . Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day (p

Techniques Used: Activity Assay, Zymography, Software, Expressing, Enzyme-linked Immunosorbent Assay

Production of MMP-2 and MMP-9 by HUVECs : Equivalent aliquots of the medium normalized to the total cell protein from the cultures of HUVECs maintained in MCDB 131 medium collected at intervals of 24 hrs were subjected to zymography ( A ) and ELISA ( C ) as described in text. Lanes 1-5 1 st -5 th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm 2 /mg protein (B) . Values given are the average of intensity measurement of 6 experiments ± SEM. The protein level expression quantitated for MMP-9 and MMP-2 by ELISA using specific antibody (C) . Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day p
Figure Legend Snippet: Production of MMP-2 and MMP-9 by HUVECs : Equivalent aliquots of the medium normalized to the total cell protein from the cultures of HUVECs maintained in MCDB 131 medium collected at intervals of 24 hrs were subjected to zymography ( A ) and ELISA ( C ) as described in text. Lanes 1-5 1 st -5 th day. The activity of MMP-9 and MMP-2 in zymogram was quantitated using Quantity One 4.5.0 software BioRad geldoc and expressed in arbitrary units of intensity/mm 2 /mg protein (B) . Values given are the average of intensity measurement of 6 experiments ± SEM. The protein level expression quantitated for MMP-9 and MMP-2 by ELISA using specific antibody (C) . Values given are the average of duplicate analysis of 5 experiments ± SEM. * Statistically significant compared to the production on first day p

Techniques Used: Zymography, Enzyme-linked Immunosorbent Assay, Activity Assay, Software, Expressing

4) Product Images from "Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirre Cells"

Article Title: Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirre Cells

Journal: BioMed Research International

doi: 10.1155/2014/412075

Genes expression profile of AFT024 cells interacted with CD34 + cells and semiquantitative RT-PCR of selected genes. The figure shows the effect of interaction between hUCB-HSCs/HPCs and AFT024-hkirre cells on the transcription of Wnt-5A, BMP4, SDF- α , and TGF- β . The AFT024-hkire cells (1.0 × 10 6 ) were cocultured with CD34 + cells (1.0 × 10 5 /well) in six-well plates for 24 hr and then washed twice to remove loosely adherent and nonadherent cells. The adherent cells were harvested and analyzed by semiquantitative RT-PCR for transcripts of the above-mentioned genes. Fold changes at 35 cycles were determined through densitometric analysis using Fluor-STM Multi-imager and Quality One 4.3.0 software (Bio-RAD) (a) expression from AFT024-hkirre cells, (b) expression from the AFT024-control cells, (c) folds of change from AFT024-hkirre group, and (d) folds of changes from control group. The data shown was a representative experiment of three reproducible experiments. (e) Cytokine (profile) expression profile was analyzed by RT-PCR. Upper panels show AFT024-hKirre cells group, and lower panels show the AFT024 control cells group.
Figure Legend Snippet: Genes expression profile of AFT024 cells interacted with CD34 + cells and semiquantitative RT-PCR of selected genes. The figure shows the effect of interaction between hUCB-HSCs/HPCs and AFT024-hkirre cells on the transcription of Wnt-5A, BMP4, SDF- α , and TGF- β . The AFT024-hkire cells (1.0 × 10 6 ) were cocultured with CD34 + cells (1.0 × 10 5 /well) in six-well plates for 24 hr and then washed twice to remove loosely adherent and nonadherent cells. The adherent cells were harvested and analyzed by semiquantitative RT-PCR for transcripts of the above-mentioned genes. Fold changes at 35 cycles were determined through densitometric analysis using Fluor-STM Multi-imager and Quality One 4.3.0 software (Bio-RAD) (a) expression from AFT024-hkirre cells, (b) expression from the AFT024-control cells, (c) folds of change from AFT024-hkirre group, and (d) folds of changes from control group. The data shown was a representative experiment of three reproducible experiments. (e) Cytokine (profile) expression profile was analyzed by RT-PCR. Upper panels show AFT024-hKirre cells group, and lower panels show the AFT024 control cells group.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Software

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    Bio-Rad imagelab software
    Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. <t>ImageLab</t> software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P
    Imagelab Software, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad electroporation cuvette
    <t>Electroporation</t> of Cas9 guide RNA complexes results in genome editing in adult polyps. (A) Partial representation of the GFP coding sequence. Target sites of sgRNAs used are shown in bold. Corresponding protospacer adjacent motifs are underlined. Arrowheads indicate cleavage sites of Cas9-sgRNPs. (B) In vitro Cas9 cleavage assay. A PCR fragment corresponding to the sequence in (A) was incubated with either Cas9 only (control) or Cas9 in combination with the sgRNAs GFPsg1-3. Presence of individual sgRNAs led to the anticipated cleavage products (GFPsg1: 104 bp + 304 bp, GFPsg2: 150 bp + 257 bp, GFPsg3: 189 bp + 219 bp). (C) Fluorescence microscopy of polyps treated with either Cas9 protein or Cas9 in combination with corresponding sgRNAs, as indicated, 7 days after electroporation. Upper panel shows an overview of the whole polyp. Superimpositions of GFP (green) and dsRed (magenta) channels are shown. A dashed rectangle indicates the area shown at high magnification in the lower panel. Arrowheads in Cas9-GFPsg1 RNP electroporated animals indicate regions with absent GFP signal. (D) Mismatch cleavage assay in Cas9 only and Cas9-GFPsg1-3 treated animals. PCR fragments of the GFP locus (A) were denatured and reannealed to allow heteroduplex formation (wildtype and mutated DNA strand). Heteroduplexes were interrogated by T7 endonuclease that cuts imperfectly matched DNA. Arrowheads indicate cleavage products in Cas9-GFPsg1 electroporated animals at 100 and 300bp. (E) Quantitative assessment of InDels in the GFP coding sequence of Cas9-GFPsg1 treated polyps. The GFP locus of treated and mock-treated animals was sequenced by Sanger sequencing following decomposition of the sequence tracks using the TIDE software ( Brinkman et al., 2014 ). The relative abundance of insertion and deletions of every length with respect to the Cas9-sgRNA RNP cutting site are shown. (F) Protein extracts from treated and mock-treated animals were analyzed using GFP-specific antibodies to assess GFP protein levels. Tubulin served as a loading control. Numbers indicate the relative amounts of GFP protein that were determined by densitometric analysis of the bands. Tubulin signals were used to normalize loading.
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    Bio-Rad version 4 4 0 software
    Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.
    Version 4 4 0 Software, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad bioplex manager software
    Multiplex cytokine analysis of ex vivo pleural fluid samples. Pleural fluid from subjects with TB (n = 27) and subjects without TB (n = 11) was assessed for 27 different cytokines using a <t>Bioplex</t> multi-cytokine analyser. Results are shown for IFN-γ, Eotaxin, IL-10, IL-13, IL6 and IP-10 levels in unstimulated fluid. Statistical analysis was performed using logistic regression analysis with Stata 3 software and multiple comparisons corrected for using Bonferonni's correction. Significance was set at p
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    Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Downregulated caveolin-1 expression serves a potential role in coronary artery spasm by inducing nitric oxide production in vitro

    doi: 10.3892/etm.2018.6646

    Figure Lengend Snippet: Western blot analysis of Cav-1 protein expression in HUVECs transfected with siCav-1(1), siCav-1(2) and siCav-1(3). GAPDH was used as a loading control. ImageLab software was used to quantify the immunoreactive band density, and GraphPad Prism version 5 software was used to generate the histogram. *P

    Article Snippet: Relative quantification of proteins was performed using ImageLab software (version 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

    Techniques: Western Blot, Expressing, Transfection, Software

    Electroporation of Cas9 guide RNA complexes results in genome editing in adult polyps. (A) Partial representation of the GFP coding sequence. Target sites of sgRNAs used are shown in bold. Corresponding protospacer adjacent motifs are underlined. Arrowheads indicate cleavage sites of Cas9-sgRNPs. (B) In vitro Cas9 cleavage assay. A PCR fragment corresponding to the sequence in (A) was incubated with either Cas9 only (control) or Cas9 in combination with the sgRNAs GFPsg1-3. Presence of individual sgRNAs led to the anticipated cleavage products (GFPsg1: 104 bp + 304 bp, GFPsg2: 150 bp + 257 bp, GFPsg3: 189 bp + 219 bp). (C) Fluorescence microscopy of polyps treated with either Cas9 protein or Cas9 in combination with corresponding sgRNAs, as indicated, 7 days after electroporation. Upper panel shows an overview of the whole polyp. Superimpositions of GFP (green) and dsRed (magenta) channels are shown. A dashed rectangle indicates the area shown at high magnification in the lower panel. Arrowheads in Cas9-GFPsg1 RNP electroporated animals indicate regions with absent GFP signal. (D) Mismatch cleavage assay in Cas9 only and Cas9-GFPsg1-3 treated animals. PCR fragments of the GFP locus (A) were denatured and reannealed to allow heteroduplex formation (wildtype and mutated DNA strand). Heteroduplexes were interrogated by T7 endonuclease that cuts imperfectly matched DNA. Arrowheads indicate cleavage products in Cas9-GFPsg1 electroporated animals at 100 and 300bp. (E) Quantitative assessment of InDels in the GFP coding sequence of Cas9-GFPsg1 treated polyps. The GFP locus of treated and mock-treated animals was sequenced by Sanger sequencing following decomposition of the sequence tracks using the TIDE software ( Brinkman et al., 2014 ). The relative abundance of insertion and deletions of every length with respect to the Cas9-sgRNA RNP cutting site are shown. (F) Protein extracts from treated and mock-treated animals were analyzed using GFP-specific antibodies to assess GFP protein levels. Tubulin served as a loading control. Numbers indicate the relative amounts of GFP protein that were determined by densitometric analysis of the bands. Tubulin signals were used to normalize loading.

    Journal: bioRxiv

    Article Title: Genetic knockdown and knockout approaches in Hydra

    doi: 10.1101/230300

    Figure Lengend Snippet: Electroporation of Cas9 guide RNA complexes results in genome editing in adult polyps. (A) Partial representation of the GFP coding sequence. Target sites of sgRNAs used are shown in bold. Corresponding protospacer adjacent motifs are underlined. Arrowheads indicate cleavage sites of Cas9-sgRNPs. (B) In vitro Cas9 cleavage assay. A PCR fragment corresponding to the sequence in (A) was incubated with either Cas9 only (control) or Cas9 in combination with the sgRNAs GFPsg1-3. Presence of individual sgRNAs led to the anticipated cleavage products (GFPsg1: 104 bp + 304 bp, GFPsg2: 150 bp + 257 bp, GFPsg3: 189 bp + 219 bp). (C) Fluorescence microscopy of polyps treated with either Cas9 protein or Cas9 in combination with corresponding sgRNAs, as indicated, 7 days after electroporation. Upper panel shows an overview of the whole polyp. Superimpositions of GFP (green) and dsRed (magenta) channels are shown. A dashed rectangle indicates the area shown at high magnification in the lower panel. Arrowheads in Cas9-GFPsg1 RNP electroporated animals indicate regions with absent GFP signal. (D) Mismatch cleavage assay in Cas9 only and Cas9-GFPsg1-3 treated animals. PCR fragments of the GFP locus (A) were denatured and reannealed to allow heteroduplex formation (wildtype and mutated DNA strand). Heteroduplexes were interrogated by T7 endonuclease that cuts imperfectly matched DNA. Arrowheads indicate cleavage products in Cas9-GFPsg1 electroporated animals at 100 and 300bp. (E) Quantitative assessment of InDels in the GFP coding sequence of Cas9-GFPsg1 treated polyps. The GFP locus of treated and mock-treated animals was sequenced by Sanger sequencing following decomposition of the sequence tracks using the TIDE software ( Brinkman et al., 2014 ). The relative abundance of insertion and deletions of every length with respect to the Cas9-sgRNA RNP cutting site are shown. (F) Protein extracts from treated and mock-treated animals were analyzed using GFP-specific antibodies to assess GFP protein levels. Tubulin served as a loading control. Numbers indicate the relative amounts of GFP protein that were determined by densitometric analysis of the bands. Tubulin signals were used to normalize loading.

    Article Snippet: 20 animals were then transferred into an electroporation cuvette (Gene Pulser / Micro Pulser Electroporation Cuvette 0.4 cm gap; Bio-Rad) and residual water was removed as completely as possible. siRNA was diluted in ddH2 O to a final concentration of 1-5 μM and 200 μL of the solution was added to each cuvette.

    Techniques: Electroporation, Sequencing, In Vitro, Cleavage Assay, Polymerase Chain Reaction, Incubation, Fluorescence, Microscopy, Software

    Electroporation of Cas9-sgRNA RNP mediates genome editing in all stem cell lineages. (A) Fluorescence microscopy of hydra strains featuring GFP expression in the ectoderm, the endoderm, the interstitial cells, and in nerve cells as indicated. Animals were electroporated with either Cas9 proteins only or with Cas9-GFPsg1 RNPs and analyzed 7 days after electroporation. Superimpositions of the GFP-(green) and the dsRed (magenta) channel are shown. The dashed rectangle in the upper panel indicates the area that is shown at high magnification in the lower panel. Arrowheads indicate regions devoid of GFP signal in the Cas9-GFPsg1 RNP-treated animals. (B) Mismatch cleavage assay of treated and mock-treated polyps from the different Hydra lines analyzed. See caption of Fig. 3 for further details. (C) Quantitative assessment of surviving animals, amounts of animals showing altered GFP expression, and genome editing efficiency obtained by TIDE analysis. Average numbers from three independent experiments are shown. (D) GFP protein levels in the electroporated animals. Western Blot analyses using anti-GFP antibodies were performed to visualize differences in GFP protein amounts. After analysis with the anti-GFP antibodies, tubulin was visualized as a loading control.

    Journal: bioRxiv

    Article Title: Genetic knockdown and knockout approaches in Hydra

    doi: 10.1101/230300

    Figure Lengend Snippet: Electroporation of Cas9-sgRNA RNP mediates genome editing in all stem cell lineages. (A) Fluorescence microscopy of hydra strains featuring GFP expression in the ectoderm, the endoderm, the interstitial cells, and in nerve cells as indicated. Animals were electroporated with either Cas9 proteins only or with Cas9-GFPsg1 RNPs and analyzed 7 days after electroporation. Superimpositions of the GFP-(green) and the dsRed (magenta) channel are shown. The dashed rectangle in the upper panel indicates the area that is shown at high magnification in the lower panel. Arrowheads indicate regions devoid of GFP signal in the Cas9-GFPsg1 RNP-treated animals. (B) Mismatch cleavage assay of treated and mock-treated polyps from the different Hydra lines analyzed. See caption of Fig. 3 for further details. (C) Quantitative assessment of surviving animals, amounts of animals showing altered GFP expression, and genome editing efficiency obtained by TIDE analysis. Average numbers from three independent experiments are shown. (D) GFP protein levels in the electroporated animals. Western Blot analyses using anti-GFP antibodies were performed to visualize differences in GFP protein amounts. After analysis with the anti-GFP antibodies, tubulin was visualized as a loading control.

    Article Snippet: 20 animals were then transferred into an electroporation cuvette (Gene Pulser / Micro Pulser Electroporation Cuvette 0.4 cm gap; Bio-Rad) and residual water was removed as completely as possible. siRNA was diluted in ddH2 O to a final concentration of 1-5 μM and 200 μL of the solution was added to each cuvette.

    Techniques: Electroporation, Fluorescence, Microscopy, Expressing, Cleavage Assay, Western Blot

    RNAi efficiency in dependence of voltage and siRNA concentration. (A) Animals pulsed at 200 V to 250 V or at a concentration of 1 μM siGFP (or less) recover within 48 hrs; polyps electroporated at 300 V or at a concentration of 5 μM siGFP do not show complete recovery. (B) Animals electroporated with siGFP exhibited a reduced fluorescence at the side of electroporation; scrambled control siRNA did not show any effects on GFP fluorescence. The silencing efficiency increases with higher voltages or application of higher siRNA concentrations. Upper panel shows whole polyps with both channels, GFP (green) and dsRed (magenta). Dashed rectangles mark the region used for magnification displayed in the lower panel. (C). The viability ranges between 94-100% for moderate electrotransfer conditions, while the application of high siRNA concentrations and high voltages results in survival decrease. (D) Quantitative analysis by RT-qPCR of whole animals confirms the reduction of GFP expression level. The degree of decrease is dependent on the stringency of the chosen electroporation conditions.

    Journal: bioRxiv

    Article Title: Genetic knockdown and knockout approaches in Hydra

    doi: 10.1101/230300

    Figure Lengend Snippet: RNAi efficiency in dependence of voltage and siRNA concentration. (A) Animals pulsed at 200 V to 250 V or at a concentration of 1 μM siGFP (or less) recover within 48 hrs; polyps electroporated at 300 V or at a concentration of 5 μM siGFP do not show complete recovery. (B) Animals electroporated with siGFP exhibited a reduced fluorescence at the side of electroporation; scrambled control siRNA did not show any effects on GFP fluorescence. The silencing efficiency increases with higher voltages or application of higher siRNA concentrations. Upper panel shows whole polyps with both channels, GFP (green) and dsRed (magenta). Dashed rectangles mark the region used for magnification displayed in the lower panel. (C). The viability ranges between 94-100% for moderate electrotransfer conditions, while the application of high siRNA concentrations and high voltages results in survival decrease. (D) Quantitative analysis by RT-qPCR of whole animals confirms the reduction of GFP expression level. The degree of decrease is dependent on the stringency of the chosen electroporation conditions.

    Article Snippet: 20 animals were then transferred into an electroporation cuvette (Gene Pulser / Micro Pulser Electroporation Cuvette 0.4 cm gap; Bio-Rad) and residual water was removed as completely as possible. siRNA was diluted in ddH2 O to a final concentration of 1-5 μM and 200 μL of the solution was added to each cuvette.

    Techniques: Concentration Assay, Fluorescence, Electroporation, Electrotransfer, Quantitative RT-PCR, Expressing

    Generation of stable GFP-mutant lines by CRISPR/Cas9 mediated genome editing. Polyps of the ecto::GFP Hydra strain (left) were electroporated with Cas9-GFPsg1 RNPs to induce genome editing at the GFP-locus. Twelve days post electroporation (middle), tissue regions devoid of GFP expression were excised and allowed to regenerated into intact polyps, which produced offspring completely lacking GFP expression. This GFP-less phenotype was stably maintained even after one year of culture (right).

    Journal: bioRxiv

    Article Title: Genetic knockdown and knockout approaches in Hydra

    doi: 10.1101/230300

    Figure Lengend Snippet: Generation of stable GFP-mutant lines by CRISPR/Cas9 mediated genome editing. Polyps of the ecto::GFP Hydra strain (left) were electroporated with Cas9-GFPsg1 RNPs to induce genome editing at the GFP-locus. Twelve days post electroporation (middle), tissue regions devoid of GFP expression were excised and allowed to regenerated into intact polyps, which produced offspring completely lacking GFP expression. This GFP-less phenotype was stably maintained even after one year of culture (right).

    Article Snippet: 20 animals were then transferred into an electroporation cuvette (Gene Pulser / Micro Pulser Electroporation Cuvette 0.4 cm gap; Bio-Rad) and residual water was removed as completely as possible. siRNA was diluted in ddH2 O to a final concentration of 1-5 μM and 200 μL of the solution was added to each cuvette.

    Techniques: Mutagenesis, CRISPR, Electroporation, Expressing, Produced, Stable Transfection

    Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.

    Journal: BMC Cell Biology

    Article Title: Degradation of the LDL receptors by PCSK9 is not mediated by a secreted protein acted upon by PCSK9 extracellularly

    doi: 10.1186/1471-2121-8-9

    Figure Lengend Snippet: Effect of nocodazole and ammonium chloride on PCSK9-mediated degradation of the LDLR . HepG2 cells were cultured in media supplemented with nocodazole (20 μg/ml) or ammonium chloride (NH 4 Cl, 10 mM) for 30 min. The media were then replaced with conditioned media from HepG2 cells transiently transfected with D374Y- PCSK9 -FLAG plasmid or with empty plasmid, already containing ammonium chloride or nocodazole, and the incubation was continued for 3 h. The conditioned media had also been added ammonium chloride or nocodazole. Cell membranes were isolated and membrane proteins equivalent to 10 μg were subjected to western blot analysis to determine the amount of LDLR. The amount of transferrin receptor (TFRC) was used as a control. Band intensities were quantified using a ChemiDoc XRS and Quantity One version 4.4.0 software. Results represent the mean (± SD) of four experiments, from which one representative western blot is shown.

    Article Snippet: Quantity One version 4.4.0 software (Bio-Rad) was used for quantification of band intensities.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Isolation, Western Blot, Software

    Multiplex cytokine analysis of ex vivo pleural fluid samples. Pleural fluid from subjects with TB (n = 27) and subjects without TB (n = 11) was assessed for 27 different cytokines using a Bioplex multi-cytokine analyser. Results are shown for IFN-γ, Eotaxin, IL-10, IL-13, IL6 and IP-10 levels in unstimulated fluid. Statistical analysis was performed using logistic regression analysis with Stata 3 software and multiple comparisons corrected for using Bonferonni's correction. Significance was set at p

    Journal: PLoS ONE

    Article Title: Highly Accurate Diagnosis of Pleural Tuberculosis by Immunological Analysis of the Pleural Effusion

    doi: 10.1371/journal.pone.0030324

    Figure Lengend Snippet: Multiplex cytokine analysis of ex vivo pleural fluid samples. Pleural fluid from subjects with TB (n = 27) and subjects without TB (n = 11) was assessed for 27 different cytokines using a Bioplex multi-cytokine analyser. Results are shown for IFN-γ, Eotaxin, IL-10, IL-13, IL6 and IP-10 levels in unstimulated fluid. Statistical analysis was performed using logistic regression analysis with Stata 3 software and multiple comparisons corrected for using Bonferonni's correction. Significance was set at p

    Article Snippet: The plate was again washed and resuspended in 125 µl of assay buffer, sealed, mixed and immediately read on the Bioplex analyser using Bioplex manager software (version 4.0; Bio-Rad, USA) and a low PMT setting.

    Techniques: Multiplex Assay, Ex Vivo, Software