Journal: PLoS ONE

Article Title: Expressional Changes in Cerebrovascular Receptors after Experimental Transient Forebrain Ischemia

doi: 10.1371/journal.pone.0041852

Figure Lengend Snippet: Photomicrographs showing immunohistochemical stainings of anterior cerebral artery (ACA) sections with antibodies against endothelin type B (ET B ) ( A and B ) or endothelin typ A (ET A ) ( D and E ) receptors. Bar graphs show quantifications of staining intensities for ET B ( C ) and ET A ( F ) receptors. Two sections from each of 6 to 11 animals were analyzed. Data are presented as mean SEM in percentages of the mean staining intensity in control-operated (sham) animals. Significant differences between sham-operated and 15 minutes ischemia rats were determined using student´s t-test. *p< 0.05.

Article Snippet: The membranes were then blocked for unspecific binding for 1 hour at room temperature in TBS-T buffer (TBS +1% Tween20 (sigma) containing 5% BSA and thereafter incubated over night at 4°C with primary antibodies: Rabbit anti-ET B (Alomone labs) diluted 1∶250, goat anti-5-HT 1B (Mybiosource) diluted 1∶250 or mouse anti-actin (Abcam) diluted 1∶15000.

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Expressional Changes in Cerebrovascular Receptors after Experimental Transient Forebrain Ischemia

doi: 10.1371/journal.pone.0041852

Figure Lengend Snippet: Representative western blots and band intensity quantifications showing endothelin type B (ET B ) protein expression in basilar artery (BA) ( A ) and in pooled middle cerebral artery (MCA) and anterior cerebral artery (ACA) tissue ( B ), and 5-hydroxotryptamin type 1B (5-HT 1B ) protein expression in BA ( C ) and in pooled MCA and ACA tissue ( D ) from control-operated (sham) rats and rats subjected to 15 minutes of transient forebrain ischemia. The band recognised by the ET B receptor antibody was approximately 48 kDa and the band recognised by the 5-HT1B receptor antibody was approximately 41 kDa. Actin was used as a loading control. Data are expressed as mean ± SEM percentages of the mean band intensity in sham-operated animals and student’s t-test was used for statistical comparison. n = 5–13. *p<0.05.

Article Snippet: The membranes were then blocked for unspecific binding for 1 hour at room temperature in TBS-T buffer (TBS +1% Tween20 (sigma) containing 5% BSA and thereafter incubated over night at 4°C with primary antibodies: Rabbit anti-ET B (Alomone labs) diluted 1∶250, goat anti-5-HT 1B (Mybiosource) diluted 1∶250 or mouse anti-actin (Abcam) diluted 1∶15000.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Expressional Changes in Cerebrovascular Receptors after Experimental Transient Forebrain Ischemia

doi: 10.1371/journal.pone.0041852

Figure Lengend Snippet: Data for endothelin type B (ET B ) and 5-hydroxotryptamin type 1B (5-HT 1B ) receptor-mediated contractile function represent E max values for the first phase (E max-1 ) of the biphasic concentration-contraction curves for anterior cerebral arteries (ACA) stimulated with endothelin-1 (ET-1) and 5-carboxamidotryptamine (5-CT), respectively. Data for ET B and 5-HT 1B protein expression levels represent receptor subtype-specific immunohistochemical staining intensities in ACAs. Neurology score data represents pooled data from grip strength and rotating pole. All data are values for ischemia-induced rats (2, 10 or 15 minutes) terminated 48 hours after ischemia in percentages of corresponding values for control-operated (sham) rats. All data are expressed as mean ± SEM.

Article Snippet: The membranes were then blocked for unspecific binding for 1 hour at room temperature in TBS-T buffer (TBS +1% Tween20 (sigma) containing 5% BSA and thereafter incubated over night at 4°C with primary antibodies: Rabbit anti-ET B (Alomone labs) diluted 1∶250, goat anti-5-HT 1B (Mybiosource) diluted 1∶250 or mouse anti-actin (Abcam) diluted 1∶15000.

Techniques: Concentration Assay, Expressing, Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Genes related to the excretion GO term and upregulated by NMDA injection and downregulated by KUS121 and KUS187 treatment.

Article Snippet: The primary antibodies were mouse anti-EDN1 antibody (1:200; Abcam) and rabbit anti-EDNRB antibody (1:100; Alomone Labs) and the secondary antibodies were biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA, USA) for EDN1 and biotinylated anti-mouse IgG (1:250; Vector Laboratories) for EDNRB.

Techniques: Injection

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: mRNA expressions of endothelin-1 ( Edn1 ), endothelin receptor type A ( Ednra ) and endothelin receptor type B ( Ednrb ) in the mouse retina and primary retinal ganglion cells (RGCs). ( a – c ) Relative mRNA expression in neural retina. Neural retina of non-treated wild-type mice (W, n = 7) and NMDA-injected (C, n = 8), NMDA-injected-KUS121-treated (K121, n = 8) and NMDA-injected-KUS187-treated (K187, n = 8) mice were analyzed. The relative expression levels of Edn1 ( a ), Ednrb ( b ) and Ednra ( c ) mRNA were analyzed using qRT-PCR. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. * p < 0.05 and ** p < 0.01, vs. W, Tukey’s honestly significant difference (HSD). NS no significant difference compared with W, Tukey’s HSD. ( d – f ) Relative mRNA expression in primary RGCs. Primary RGCs isolated from 3-day-old rats by two-step immunopanning were cultured with or without KUS121 (50 µM) for 2 h and then with or without KUS121 and with or without NMDA (500 µM) for another 4 h. The relative expression levels of Edn1 ( d ), Ednrb ( e ) and Ednra ( f ) mRNA were analyzed using qRT-PCR. (-): without KUS121 without NMDA n = 3, C: with NMDA without KUS121, n = 3, K121: with NMDA with KUS121, n = 3, respectively. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. ** p < 0.01, control vs. KUS121, NS no significant difference compared with (-), Tukey’s HSD.

Article Snippet: The primary antibodies were mouse anti-EDN1 antibody (1:200; Abcam) and rabbit anti-EDNRB antibody (1:100; Alomone Labs) and the secondary antibodies were biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA, USA) for EDN1 and biotinylated anti-mouse IgG (1:250; Vector Laboratories) for EDNRB.

Techniques: Expressing, Injection, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Protein expression of endothelin-1 (EDN1) and endothelin receptor type B (EDNRB) in retinal tissue. ( a ) The retina of non-treated wild-type (labeled ‘‘W’’), NMDA-injected (control, labeled ‘‘C’’), or NMDA-injected-KUS121-treated mice (labeled ‘‘K’’) was analyzed by western blotting using an anti-EDN1 antibody. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. a. ( b ) Comparison of EDN1 expression shown as ratios to actin ( n = 5, for all treatments). ** p < 0.01, vs. W, Tukey’s HSD. ( c – f ) Vertical sections of non-treated wild-type, NMDA-injected, NMDA-injected-KUS121-treated and NMDA-injected-KUS187-treated mice retinae were stained with an anti-EDN1 antibody or anti-EDNRB antibody. ( c , d ) Staining intensities of RGC layers with anti-EDN1 ( c ) or anti-EDNRB ( d ) antibody. The staining intensity of the RGC layer at distances of 400–800 μm from the optic nerve head was analyzed using BZ II Analyzer software. W: non-treated wild-type, C: NMDA-injected, K121: NMDA-injected-KUS121-treated, K187: NMDA-injected-KUS187-treated. ( n = 3, for C and K121, n = 2, for W and K187) NS no significant difference compared with W, Tukey’s HSD. ( e,f ) Vertical sections of non-treated wild-type (WT), NMDA-injected (control), NMDA-injected-KUS121-treated (K121) and NMDA-injected-KUS187-treated mice (K187) retinae. The black bar represents 100 µm. GCL ganglion cell layer, IPL inner plexiform layer.

Article Snippet: The primary antibodies were mouse anti-EDN1 antibody (1:200; Abcam) and rabbit anti-EDNRB antibody (1:100; Alomone Labs) and the secondary antibodies were biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA, USA) for EDN1 and biotinylated anti-mouse IgG (1:250; Vector Laboratories) for EDNRB.

Techniques: Expressing, Labeling, Injection, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: EDN1 and EDNRB protein expression and cell viability in 661W cultured cells under glucose-free conditions. ( a – d ) The relative amount of live cells was measured using WST-8 following 48 h treatment with DMEM/high glucose media or DMEM/glucose-free media with or without KUS121 (100 µM). ( a ) Quantitative analysis of live cells ( n = 3, for all treatments). 661W cells were cultured with high glucose [labeled ‘‘(-)’’] or treated in glucose-free media with (labeled ‘‘K121’’) or without (control, labeled ‘‘C’’) KUS121. ( b – d ) Representative images of 661W cells cultured under each condition. The black bar represents 50 µm. ( e – h ) Protein expression of EDN1 ( e , g ) and EDNRB ( f , h ) in 661W cells was analyzed by western blotting. 661W cells were cultured with high glucose media [labeled ‘‘(-)’’] or with glucose-free media with (labeled ‘‘K’’) or without (control, labeled ‘‘C’’) KUS121 for 24 h before the analysis. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. b,c. Relative expression of EDN1 ( g ) and EDNRB ( h ) was shown as a ratio to actin ( n = 4, for both treatments). * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the control (in a and g ) or to the cells cultured in high glucose media (in h ), Tukey’s HSD.

Article Snippet: The primary antibodies were mouse anti-EDN1 antibody (1:200; Abcam) and rabbit anti-EDNRB antibody (1:100; Alomone Labs) and the secondary antibodies were biotinylated anti-mouse IgG (1:250; Vector Laboratories, Burlingame, CA, USA) for EDN1 and biotinylated anti-mouse IgG (1:250; Vector Laboratories) for EDNRB.

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Genes related to the excretion GO term and upregulated by NMDA injection and downregulated by KUS121 and KUS187 treatment.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Injection

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: mRNA expressions of endothelin-1 ( Edn1 ), endothelin receptor type A ( Ednra ) and endothelin receptor type B ( Ednrb ) in the mouse retina and primary retinal ganglion cells (RGCs). ( a – c ) Relative mRNA expression in neural retina. Neural retina of non-treated wild-type mice (W, n = 7) and NMDA-injected (C, n = 8), NMDA-injected-KUS121-treated (K121, n = 8) and NMDA-injected-KUS187-treated (K187, n = 8) mice were analyzed. The relative expression levels of Edn1 ( a ), Ednrb ( b ) and Ednra ( c ) mRNA were analyzed using qRT-PCR. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. * p < 0.05 and ** p < 0.01, vs. W, Tukey’s honestly significant difference (HSD). NS no significant difference compared with W, Tukey’s HSD. ( d – f ) Relative mRNA expression in primary RGCs. Primary RGCs isolated from 3-day-old rats by two-step immunopanning were cultured with or without KUS121 (50 µM) for 2 h and then with or without KUS121 and with or without NMDA (500 µM) for another 4 h. The relative expression levels of Edn1 ( d ), Ednrb ( e ) and Ednra ( f ) mRNA were analyzed using qRT-PCR. (-): without KUS121 without NMDA n = 3, C: with NMDA without KUS121, n = 3, K121: with NMDA with KUS121, n = 3, respectively. The ratios of mRNA expression of each gene to that of glyceraldehyde-3-phosphate dehydrogenase were calculated. ** p < 0.01, control vs. KUS121, NS no significant difference compared with (-), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Injection, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: Protein expression of endothelin-1 (EDN1) and endothelin receptor type B (EDNRB) in retinal tissue. ( a ) The retina of non-treated wild-type (labeled ‘‘W’’), NMDA-injected (control, labeled ‘‘C’’), or NMDA-injected-KUS121-treated mice (labeled ‘‘K’’) was analyzed by western blotting using an anti-EDN1 antibody. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. a. ( b ) Comparison of EDN1 expression shown as ratios to actin ( n = 5, for all treatments). ** p < 0.01, vs. W, Tukey’s HSD. ( c – f ) Vertical sections of non-treated wild-type, NMDA-injected, NMDA-injected-KUS121-treated and NMDA-injected-KUS187-treated mice retinae were stained with an anti-EDN1 antibody or anti-EDNRB antibody. ( c , d ) Staining intensities of RGC layers with anti-EDN1 ( c ) or anti-EDNRB ( d ) antibody. The staining intensity of the RGC layer at distances of 400–800 μm from the optic nerve head was analyzed using BZ II Analyzer software. W: non-treated wild-type, C: NMDA-injected, K121: NMDA-injected-KUS121-treated, K187: NMDA-injected-KUS187-treated. ( n = 3, for C and K121, n = 2, for W and K187) NS no significant difference compared with W, Tukey’s HSD. ( e,f ) Vertical sections of non-treated wild-type (WT), NMDA-injected (control), NMDA-injected-KUS121-treated (K121) and NMDA-injected-KUS187-treated mice (K187) retinae. The black bar represents 100 µm. GCL ganglion cell layer, IPL inner plexiform layer.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Labeling, Injection, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Involvement of endothelins in neuroprotection of valosin-containing protein modulators against retinal ganglion cell damage

doi: 10.1038/s41598-022-20497-w

Figure Lengend Snippet: EDN1 and EDNRB protein expression and cell viability in 661W cultured cells under glucose-free conditions. ( a – d ) The relative amount of live cells was measured using WST-8 following 48 h treatment with DMEM/high glucose media or DMEM/glucose-free media with or without KUS121 (100 µM). ( a ) Quantitative analysis of live cells ( n = 3, for all treatments). 661W cells were cultured with high glucose [labeled ‘‘(-)’’] or treated in glucose-free media with (labeled ‘‘K121’’) or without (control, labeled ‘‘C’’) KUS121. ( b – d ) Representative images of 661W cells cultured under each condition. The black bar represents 50 µm. ( e – h ) Protein expression of EDN1 ( e , g ) and EDNRB ( f , h ) in 661W cells was analyzed by western blotting. 661W cells were cultured with high glucose media [labeled ‘‘(-)’’] or with glucose-free media with (labeled ‘‘K’’) or without (control, labeled ‘‘C’’) KUS121 for 24 h before the analysis. Actin was used as a loading control. Complete scans of western blots are shown in Supplementary Fig. b,c. Relative expression of EDN1 ( g ) and EDNRB ( h ) was shown as a ratio to actin ( n = 4, for both treatments). * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with the control (in a and g ) or to the cells cultured in high glucose media (in h ), Tukey’s HSD.

Article Snippet: The primary antibodies used to probe the blots were mouse anti-EDN1 (1:400; Abcam, Cambridge, UK), rabbit anti-EDNRB (1:400; Alomone Labs, Jerusalem, Israel), rabbit anti-AKT (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AKT (1:2000, Cell Signaling Technology), rabbit anti-ERK (1:20,000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-phospho-ERK (1:1000, Cell Signaling Technology) and anti-actin (1:5000; Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: PLoS ONE

Article Title: Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ET B receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

doi: 10.1371/journal.pone.0174119

Figure Lengend Snippet: Concentration-response curves show the effect of increasing concentrations of Sarafotoxin 6c (S6c) in the left anterior descending artery (LAD) upstream of the ligature, LAD downstream of the ligature, and non-ischemic septal coronary artery (SCA) after treatment with (A) IR and vehicle or (B) U0126. The results are shown as mean values ± SEM (n = 6–7), *** P < 0.001; two-way ANOVA with repeated measurements; LAD downstream vs. upstream. Concentration-response curves show the effect of increasing concentrations of endothelin-1 (ET-1) after ET B receptor desensitization and BQ788 treatment in the LAD upstream of the ligature, LAD downstream of the ligature, and non-ischemic SCA after treatment with (C) the vehicle or (D) U0126. The results are shown as mean values ± SEM (n = 4–7), *** P < 0.001; two-way ANOVA with repeated measurements; LAD downstream vs. upstream.

Article Snippet: Membranes were subsequently blocked for 1 h in 2% ECL Prime Blocking Agent (GE Healthcare Life Sciences, Brøndby, Denmark) in TBS-T (TBS plus 0.1% Tween 20) before incubation overnight at 4°C on a rotor with the following primary antibodies diluted in TBS-T containing 0.02% sodium azide: polyclonal rabbit-anti-endothelin ET B receptor antibody (#AER-002, Alomone Laboratories, Israel 1:200); monoclonal mouse-anti-ET-1 antibody (#ab2786, Abcam, 1:1000); polyclonal rabbit-anti-endothelin ET A receptor antibody (#9780, Sigma, 1:1000); rabbit monoclonal anti-total-ERK antibody (#9101, Cell Signaling, 1:2000); or mouse monoclonal anti-pERK antibody (#9107, Cell Signaling, 1:1000).

Techniques: Concentration Assay

Journal: F1000Research

Article Title: A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions

doi: 10.12688/f1000research.7142.2

Figure Lengend Snippet: Antibodies.

Article Snippet: EDNRB Rabbit , Primary, indirect IF , 8μg/ml , Alomone labs #AER-002.

Techniques:

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Effects of human cytomegalovirus (HCMV) on vascular endothelial and smooth muscle cells. The graphic depicts relative expression of endothelin receptor type B (ET B R) with indicated multiplicity of infection (moi) of HCMV as assayed by quantitative TaqMan polymerase chain reaction (PCR) at indicated days postinfection (dpi) for endothelial cells (human umbilical vein cells [HUVECs]; A.i) or smooth muscle cells (SMCs) (human pulmonary arterial SMCs [hPASMCs], A.ii) and human aortic SMCs [hSMCs], A.iii). The corresponding infection levels in HCMV-infected HUVECs (A.iv), hPASMCs (A.v) and hSMCs (A.vi), as assessed by quantitative TaqMan PCR are shown. The effects of UV-irradiated HCMV on the ET B R mRNA level in hPASMC (A.vii) and hSMC (A.viii) are illustrated. The uninfected cells at the indicated time course were used as a calibrator for relative quantification using the comparative 2 −ΔΔCt method with β 2 -microglobulin as an endogenous control (biological replicates for A.i-A.vi [n = 4] and A.vii and A.viii [n = 3] with technical duplicates for each PCR; values are mean ± standard deviation [SD]). Representative immunofluorescence staining on HCMV-infected endothelial cells (HUVECs; B.i) and SMCs (hPASMC, B.ii and hSMC, B.iii) with indicated MOI at 3 dpi. Arrows in B.i-B.iii depict HCMV-infected cells without up-regulation of ET B R. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI], blue), and the immunoreactivity of ET B R or HCMV immediate-early (IE) was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively (biological replicates, n = 3). The corresponding quantitative results are shown for HCMV-infected HUVECs (B.iv), hPASMC (B.v) and hSMC (B.vi), by mean florescent intensity expressed as pixel sum as analyzed with Leica Application Suite Advanced Fluorescence software. At least 5 different areas of interest were quantified (values are mean ± SD; P < .05 was considered to be statistically significant; * P = .01–.05; ** P = .001–.01; *** P < .001; n.s = not significant).

Article Snippet: For dual staining, cells were incubated with rabbit anti-ET B R (1:200) (AER-002; Alomone Labs) for 30 minutes at room temperature and then washed and fixed with Reagent A followed by permeabilization with Reagent B (Molecular Probes; Invitrogen, Life Technologies) along with primary mouse anti-IE according to the manufacturer's protocol.

Techniques: Expressing, Infection, Polymerase Chain Reaction, Irradiation, Standard Deviation, Immunofluorescence, Staining, Fluorescence, Software

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Human cytomegalovirus (HCMV) infection alters endothelin receptor type B (ET B R) cellular distribution, induces surface expression of ET B R and its effect on endothelin receptor type A (ET A R). (A) Representative immunofluorescence staining on HCMV-infected endothelial cells at 3 days postinfection to illustrate the subcellular distribution of ET B R in uninfected and infected cells. Arrow shows increased ET B R protein expression in the perinuclear region. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of ET B R or HCMV immediate-early (IE) was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively (biological replicates, n = 3). (B) Representative immunofluorescence staining of ET B R and giantin, a Golgi marker (top panel), or PDI, an endoplasmic reticulum marker (bottom panel). Nucleus was stained with DAPI (blue). Flow cytometry analysis on the effects of HCMV-infected endothelial cells (human umbilical vein cells [HUVECs]) or smooth muscle cells (SMCs) (human pulmonary arterial SMCs [hPASMCs] and human aortic SMCs [hSMCs]) on ET B R at indicated days postinfection (dpi). (C) The effects of different multiplicity of infection (moi) of HCMV on ET B R surface expression at 1, 3, and 5 dpi (left panel) in HUVECs. The HCMV IE is shown on the right panel. Y-axis denotes log intensity of ET B R (left panel) or IE (right panel), whereas x-axis is side scatter in linear scale (SS Lin). (D) The effects of ultraviolet (UV)-irradiated HCMV on ET B R surface expression in HUVECs. Little or no IE was detected with the UV treatment (bottom row). (E) The effects of UV-irradiated HCMV (middle row) or HCMV (bottom row) on ET B R surface expression in SMCs, hPASMCs (left column) and hSMCs (right column) at 5 dpi. Y-axis denotes log intensity of ET B R (left panel), whereas x-axis is SS Lin. The effects of HCMV on ET A R mRNA level in smooth muscle cells, hPASMC (F) and hSMC (G) (MOI; biological replicates, n = 3 with technical duplicates for each polymerase chain reaction; values are mean ± standard deviation; P < .05 was considered to be statistically significant; * P = .01–.05; ** P = .001–.01; *** P < .001).

Article Snippet: For dual staining, cells were incubated with rabbit anti-ET B R (1:200) (AER-002; Alomone Labs) for 30 minutes at room temperature and then washed and fixed with Reagent A followed by permeabilization with Reagent B (Molecular Probes; Invitrogen, Life Technologies) along with primary mouse anti-IE according to the manufacturer's protocol.

Techniques: Infection, Expressing, Immunofluorescence, Staining, Marker, Flow Cytometry, Irradiation, Polymerase Chain Reaction, Standard Deviation

Journal: Open Forum Infectious Diseases

Article Title: Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

doi: 10.1093/ofid/ofv155

Figure Lengend Snippet: Human cytomegalovirus (HCMV) antigens are closely associated with endothelin receptor type B (ET B R) in human carotid atherosclerotic plaques. Representative photomicrographs of immunohistochemistry and immunofluorescence (IF) staining on frozen human carotid atherosclerotic plaques. (A) Immunohistochemistry staining to show colocalization of HCMV immediate-early (IE) and ET B R in Von Willebrand factor (vWF)-positive cells as revealed by chromogen diaminobenzidine, brown products in a boxed area with consecutive sections (left column; right column is a higher magnification of the boxed areas). (B) The IF staining on a human carotid atherosclerotic plaque to illustrate the colocalization of HCMV-IE with ET B R in fibroblast-like cells. Nucleus was stained with 4′-6-diamidino-2-phenylindole ([DAPI] blue), and the immunoreactivity of HCMV-IE or ET B R was revealed with Alexa Fluor 594 (red) or Alexa Fluor 488 (green), respectively. Twenty human carotid atherosclerotic plaques were examined.

Article Snippet: For dual staining, cells were incubated with rabbit anti-ET B R (1:200) (AER-002; Alomone Labs) for 30 minutes at room temperature and then washed and fixed with Reagent A followed by permeabilization with Reagent B (Molecular Probes; Invitrogen, Life Technologies) along with primary mouse anti-IE according to the manufacturer's protocol.

Techniques: Immunohistochemistry, Immunofluorescence, Staining