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: Figure 3. 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. S2a. (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, Control, Western Blot, Comparison, Staining, Software
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![Figure 4. 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. S2b,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.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1250/pm36171250/pm36171250__page6_image1.jpg)