Human Alkaline Phosphatase/ALPL Antibody

Detection of Alkaline Phosphatase/ALPL by Western Blot

Analysis of the control and CAVD aortic valve samples (n = 5 and 5, respectively). (A) Western blotting for CDK1 and ALP proteins on valve tissues. (B) Semi-quantitative analysis of protein expression. (C) Immunofluorescence staining of CDK1 and Ki-67 on valve tissues. (D) Semi-quantitative analysis of fluorescence intensity. (E) PCR test for cell proliferation genes on valve tissues. (*) p < 0.05 indicates a significant difference. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35571082), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

Analysis of the control and CAVD aortic valve samples (n = 5 and 5, respectively). (A) Western blotting for CDK1 and ALP proteins on valve tissues. (B) Semi-quantitative analysis of protein expression. (C) Immunofluorescence staining of CDK1 and Ki-67 on valve tissues. (D) Semi-quantitative analysis of fluorescence intensity. (E) PCR test for cell proliferation genes on valve tissues. (*) p < 0.05 indicates a significant difference. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35571082), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

CCND1 participates in the osteogenic differentiation of VICs following OM. A–F) VICs were transfected with CCND1 siRNA or scrambled siRNA, and then stimulated with OM for 7 days. Immunoblot analysis of CCND1, ALP, Runx2 P53, and P21 expression in VICs from indicated groups (n = 3, each group). Bar plots showing the semiquantitative analysis of indicated genes expression. G,H) With OM induction for 7 days, representative ALP staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. I–K) With OM induction for 21 days, representative Alizarin red staining showed the calcific nodules in VICs from indicated groups (n = 3, each group). Scale bar 50 µm. L–M) Representative SA‐ beta ‐gal staining of VICs from indicated groups (n = 3, each group). Bar plot showing the percentage of SA‐ beta ‐gal staining positive cells. Scale bar 50 µm. Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

Morusin attenuates VIC calcification by activating Nrf2 signaling pathway. A–D) ML385 was used to inhibit the activation of Nrf2 in VICs. Representative immunoblot images and quantification of the levels of ALP, Runx2, and P21 in VICs from indicated groups (n = 3, each group). E,F) With OM induction for 7 days, representative ALP staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. G–I) With OM induction for 21 days, representative Alizarin red staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. Data are means ± SD. **p < 0.01; ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Immunohistochemistry

Morusin inhibits OM‐induced osteogenic differentiation of VICs. A) Immunoblot analysis of Runx2 and ALP expression in VICs from indicated groups (n = 3, each group). B) Bar plot showing the fold change of Runx2 expression over control. C) Bar plot showing the fold change of ALP expression over control. D) Immunofluorescent staining of ALP (green), Runx2 (red), and DAPI (blue) in the VICs from indicated groups. Scale bar 50 µm. E,F) With OM induction for 7 days, representative ALP staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. G–I) With OM induction for 21 days, representative Alizarin red staining showed the calcific nodules in VICs from indicated groups (n = 3, each group). Scale bar 50 µm. J) With OM induction for 21 days, representative Von Kossa and Alizarin Red staining of aortic valve leaflets. K) Bar plot showing the percentage of Von Kossa positive staining area of indicated groups (n = 3, each group). L) Bar plot showing the percentage of Alizarin Red positive staining area of indicated groups (n = 3, each group). Data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

Morusin inhibits OM‐induced osteogenic differentiation of VICs. A) Immunoblot analysis of Runx2 and ALP expression in VICs from indicated groups (n = 3, each group). B) Bar plot showing the fold change of Runx2 expression over control. C) Bar plot showing the fold change of ALP expression over control. D) Immunofluorescent staining of ALP (green), Runx2 (red), and DAPI (blue) in the VICs from indicated groups. Scale bar 50 µm. E,F) With OM induction for 7 days, representative ALP staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. G–I) With OM induction for 21 days, representative Alizarin red staining showed the calcific nodules in VICs from indicated groups (n = 3, each group). Scale bar 50 µm. J) With OM induction for 21 days, representative Von Kossa and Alizarin Red staining of aortic valve leaflets. K) Bar plot showing the percentage of Von Kossa positive staining area of indicated groups (n = 3, each group). L) Bar plot showing the percentage of Alizarin Red positive staining area of indicated groups (n = 3, each group). Data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Immunocytochemistry/ Immunofluorescence

Morusin inhibits OM‐induced osteogenic differentiation of VICs. A) Immunoblot analysis of Runx2 and ALP expression in VICs from indicated groups (n = 3, each group). B) Bar plot showing the fold change of Runx2 expression over control. C) Bar plot showing the fold change of ALP expression over control. D) Immunofluorescent staining of ALP (green), Runx2 (red), and DAPI (blue) in the VICs from indicated groups. Scale bar 50 µm. E,F) With OM induction for 7 days, representative ALP staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. G–I) With OM induction for 21 days, representative Alizarin red staining showed the calcific nodules in VICs from indicated groups (n = 3, each group). Scale bar 50 µm. J) With OM induction for 21 days, representative Von Kossa and Alizarin Red staining of aortic valve leaflets. K) Bar plot showing the percentage of Von Kossa positive staining area of indicated groups (n = 3, each group). L) Bar plot showing the percentage of Alizarin Red positive staining area of indicated groups (n = 3, each group). Data are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

Morusin attenuates VIC calcification depending on Trim25. A,B) VICs were transfected with CCND1 siRNA or scrambled siRNA, immunoblot analysis of Trim25 expression in VICs from indicated groups (n = 3, each group). Bar plots showing the semiquantitative analysis of Trim25 expression. C) VICs were transfected with Trim25 siRNA or scrambled siRNA. Co‐IP analysis of the Keap1 ubiquitination level in VICs from indicated groups (n = 3, each group). D–M) Representative immunoblot images and quantification of the levels of Trim25, ALP, Runx2, and P21 in VICs from indicated groups (n = 3, each group). N,S) VICs were transfected with Trim25 siRNA or scrambled siRNA, representative ALP staining of VICs from indicated groups (n = 3, each group). P,U) VICs were transfected with Trim25 siRNA or scrambled siRNA, representative Alizarin red staining of VICs from indicated groups (n = 3, each group). Scale bar 50 µm. Data are means ± SD. NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA with Tukey's multiple comparisons test). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/38502885), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Alkaline Phosphatase/ALPL by Western Blot

Analyses of TNAP expression and activity by immunofluorescence staining, Western blot, and enzyme activity assay. Cell analyses were performed in five different ALPLtg PDL-hTERT cell lines, the wt line and control PDL-hTERT that did not undergo genome editing. (A) TNAP expression (green) was detected by immunofluorescence after nuclear counterstaining with DAPI (blue). Representative images are shown. Scale bars = 50 µm. (B) Quantification of TNAP signals in IF images were done by corrected total cell fluorescence (CTCF) for ten random cells per image, and five images per cell line (n = 50). (C) Specific TNAP activity was measured by a CSPD assay confirming the spontaneous activity of the expressed protein. N = 4 per condition. (D–F) TNAP expression level and the expression of the mitochondrial protein TOMM20 in total cell lysates were determined semi-quantitatively by Western blot analysis in relation to the housekeeping protein beta -actin/ACTB. A lysate of a TNAP overexpressing cell line (ALPL over) was used as positive control. A representative immunoblot is shown in (C). White pixels indicate signal oversaturation. Quantification results in E and F are presented as mean ± SEM, N = 3 per condition. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/40530336), licensed under a CC-BY license. Not internally tested by R&D Systems.