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: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 2 – Expression of immunoreactive ETA and ETB receptors in fluorogold-labeled trigeminal ganglion neurons. Animals received fluorogold (FG, 4% solution) injected s.c. into the upper lip. After 5–7 days, FG-injected rats were sacrificed and both TG removed and processed for ETA and ETB receptor immunohistochemistry. In panel A, images on the right depict FG-labeled TG neurons; middle images show ETA and ETB receptor staining and images on the left show expression of ETA and ETB receptors on some FG positive neurons (arrows on merged images). The asterisk indicates ETB receptor expression on satellite glial cells. Tissue sections incubated in the absence of primary or secondary antibodies did not display any fluorescence (not shown). Images are representative of at least 3 sections from each ganglion, taken from 3 to 4 distinct animals. Panel B illustrates the size–frequency histogram of ETA and ETB positive neurons within the entire trigeminal ganglion (n=1460 neurons).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Labeling, Injection, Immunohistochemistry, Staining, Incubation, Fluorescence

Journal: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 3 – Co-localization of ETA and ETB receptors with TRPV1. Sections were labeled for TRPV1 receptors (in red, left images) and ETA or ETB receptors (ETAR and ETBR, in green). Images on the right depict co-localization of TRPV1 receptors with ETAR and ETBR (arrows on merged images). Tissue sections incubated in the absence of primary or secondary antibodies did not display any fluorescence (not shown). Images are representative of at least 3 sections from each ganglion taken from 3 to 4 distinct animals. Panel B illustrates the percentage of neurons immunoreactive for TRPV1, ETA (ETAR) and ETB receptors (ETBR), as well as the percentage of TRPV1 positive neurons that also express ETAR and ETBR (n=1102 neurons).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Labeling, Incubation, Fluorescence

Journal: Brain research

Article Title: Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

doi: 10.1016/j.brainres.2010.04.075

Figure Lengend Snippet: Fig. 7 – Influence of ETA and ETB receptor antagonists on ET-1-induced nociceptive responses. BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, each at 10 nmol/site) were administered 30 min before ipsilateral ET-1 (10 pmol/site) injection into the upper lip (A) or TMJ (B). Each value represents the mean±s.e.m. of the total facial grooming time (A) or the sum of nociceptive behaviors (B) recorded from 6 to 8 animals. Asterisks denote P≤0.05 relative to the PBS plus ET-1-injected group (ANOVA followed by Bonferroni's test).

Article Snippet: For double-labeling experiments, slides were incubated simultaneously with the anti-ETA or anti-ETB receptor antibody and goat anti-TRPV1 receptor antibody (1:200; Santa Cruz Biotechnology, CA, USA).

Techniques: Injection

Journal: BMC Neuroscience

Article Title: Upregulation of the endothelin A (ET A ) receptor and its association with neurodegeneration in a rodent model of glaucoma

doi: 10.1186/s12868-017-0346-3

Figure Lengend Snippet: Overexpression of the ET A receptor can increase in ET B receptor expression. a Representative images. Immunoblot showing the expression of ET A and ET B receptors in the plasma membrane fraction of 661W stable clones overexpressing the ET A receptor. b Immunoblot densitometry showing fold change in protein expression of both ET A and ET B receptors in 661W cells overexpressing the ET A receptor compared to empty vector cells. Protein normalized to calnexin expression. Bars represent mean ± SEM (n = 5). Asterisks indicated statistical significance *p < 0.05 by student’s t-test

Article Snippet: Primary antibodies used to probe blots were rabbit anti-ET A (1:1000; Sigma), rabbit anti-ET B (1:10,000, Antibody Research Corporation), rabbit anti-Calnexin (1:1000, Cell Signaling) and mouse calnexin (1:1000, Cell Signaling).

Techniques: Over Expression, Expressing, Western Blot, Clone Assay, Plasmid Preparation

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: ERK1/2 inhibition attenuates cerebral blood flow reduction and abolishes ET(B) and 5-HT(1B) receptor upregulation after subarachnoid hemorrhage in rat.

doi: 10.1038/sj.jcbfm.9600236

Figure Lengend Snippet: Figure 4 Effect of treatment with ERK1/2 inhibitor SB386023-b in cerebral arteries on the mRNA levels of 5-HT1B and ETB receptors after experimental induced SAH in rats. (A) ETB and (B) 5-HT1B. There are upregulations of the ETB and 5-HT1B receptor mRNA levels in the SAH compared with the sham- operated rats. Treatment with SB386023-b prevented the upregulations. Data were obtained by real-time PCR and are expressed as mean7s.e.m. values relative to EF-1 mRNA levels, n = 7 to 10, *Pr0.05.

Article Snippet: The first antibodies used were rabbit antihuman ETB (IBL, Stockholm, Sweden, 16207), diluted 1:400, goat anti-mouse 5-HT1B (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, sc-1461), diluted 1:100, mouse anti-rat CD31 (Serotec, Hamar, Norway, MCA1746), diluted 1:200, and mouse anti-rat smooth muscle actin (Serotec, MCA1905T), diluted 1:100.

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism

Article Title: ERK1/2 inhibition attenuates cerebral blood flow reduction and abolishes ET(B) and 5-HT(1B) receptor upregulation after subarachnoid hemorrhage in rat.

doi: 10.1038/sj.jcbfm.9600236

Figure Lengend Snippet: Figure 5 Sections from the BA showing ETB and 5-HT1B immunoreactivity in the smooth muscle cell layer. (A) ETB; sham, (B) ETB; SAH, (C) ETB; SAH treated with SB386023-b, (D) 5-HT1B; sham, (E) 5-HT1B; SAH, (F) 5-HT1B; SAH treated with SB386023-b. There are increased expressions of the ETB and 5-HT1B receptor protein levels in the SAH compared with the sham operated rats. Treatment with SB386023-b prevented the increased protein expression in the smooth muscle cells. Data were obtained with confocal microscopy.

Article Snippet: The first antibodies used were rabbit antihuman ETB (IBL, Stockholm, Sweden, 16207), diluted 1:400, goat anti-mouse 5-HT1B (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, sc-1461), diluted 1:100, mouse anti-rat CD31 (Serotec, Hamar, Norway, MCA1746), diluted 1:200, and mouse anti-rat smooth muscle actin (Serotec, MCA1905T), diluted 1:100.

Techniques: Expressing, Confocal Microscopy