plpp3 antibody  (Novus Biologicals)

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Expression of PLPP3 is reduced in CAVD. (A) Transcriptome of PLPPs in CTL (n = 12) and CAVD specimens (n = 12), the number in parentheses following the gene name represents the relative change in expression (*indicates P < 0.05). (B) mRNA levels for PLPP3 in CTL (n = 29) and CAVD (n = 39) (P < 0.0001). (C) Western blotting for PLPP3 showing a representative experiment and the quantification in CTL (n = 8) and CAVD (n = 9) (P = 0.01). (D) PLPP enzyme activity in CTL (n = 8) and CAVD (n = 8) (P = 0.03). (E) LysoPA relative levels according to PLPP3 mRNA levels (divided at median) (n = 10) (P = 0.007). (F) PLPP3 mRNA levels in pathologic aortic valves (CAVD) according to the state of mineralization (n = 7) (P = 0.007). G) PLPP3 mRNA levels in VICs isolated from CTL (n = 5) and CAVD (n = 5) (P = 0.004). Values are mean ± SEM, (B) Student t-test, (C–G) Wilcoxon-Mann-Whitney statistical analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Western Blot, Activity Assay, Isolation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: 5meC level at cg02468627 inversely correlates with PLPP3 expression. (A) Genomic coordinates (hg19) of CpG sites (open circles) and their relationships with PLPP3 expression represented as –log10 (P-value). (B) Correlation analysis between the expression of PLPP3 and CpG methylation level (cg02468627) (n = 21) in CAVD (r = −0.51, P = 0.01). Bisulphite pyrosequencing of cg02468627 in CTL (n = 24) and CAVD (n = 43) (P < 0.0001) (C), and correlation analysis between methylation of cg02468627 with PLPP3 expression (n = 28) (r = −0.48, P = 0.009) (D). (E) Bisulphite pyrosequencing levels in pathologic aortic valves according to the state of mineralization (n = 8) (P = 0.03). Values are mean ± S.E.M.; (A) linear regression model, (B and D) Pearson’s coefficient, (C) student t-tests, (E) Wilcoxon-Mann-Whitney analysis; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, CpG Methylation Assay, Methylation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intron 1 of PLPP3 contains an enhancer. (A) UCSC browser image (hg19) showing cg02468627 in highly conserved region containing a MIR3 sequence. (B–D) H3K4me1 (CTL n = 5, CAVD n = 5) (P = 0.42) (B), H3K4me3 (CTL n = 5, CAVD n = 5) (P = 0.15) (C), and H3K27me3 (CTL n = 5, CAVD n = 6) (P = 0.017) (D) levels at MIR3 locus (determined by quantitative ChIP). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Sequencing, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intronic enhancer regulates PLPP3 expression. (A) Reporter assay constructions and activities (HEK293T) (n = 5) (P = 0.007). (B) Scheme showing dCas9-DNMT and dCas9-ΔDNMT for epigenome editing. C) Bisulphite pyrosequencing of intronic enhancer performed in HEK293T after epigenome editing (n = 5) (P = 0.03). (D) Reporter assay in response to epigenome editing (HEK293T) (n = 6) (P = 0.03). (E) Expression of PLPP3 in VICs in response to epigenome editing (n = 5) (P = 0.03). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Reporter Assay, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Lower expression of PLPP3 enhances the osteogenic transition of VICs. (A) siRNA-mediated knockdown on mRNA (n = 5) (P = 0.007) and protein levels. (B–D) PLPP3 siRNA on lysoPA-mediated gene expression of BMP2 (n = 11) (pAnova < 0.0001) (B), RUNX2 (n = 11) (pAnova < 0.0001) (C), and BGLAP (n = 9) (P < 0.0001) (D). (E and F) PLPP3 knockdown on lysoPA-induced mineralization of VIC cultures (n = 5) (day 7) (P = 0.0001) (E), and ALP activity (n = 7) (day 7) (P < 0.0001) (F). (G) Proposed working model showing that EZH2 (enzyme component of the PRC2 complex that increases H3K27me3) may associate with DNMT3 (de novo methylation of CpG) and promote silencing of PLPP3 intronic enhancer during the mineralization of the AV. Values are mean ± S.E.M.; (A) Wilcoxon-Mann-Whitney analysis, (B and C) Anova, post-hoc Tukey, (D–F) Kruskal-Wallis, post-hoc Steel; *P < 0.05; LysoPA: 10 µM; PO4 is mineralizing medium (PO4 2 mM); percentage of calcium in (E) is indicated and represents a surrogate of mineralization (hydroxyapatite of calcium) that is deposited by VICs.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Knockdown, Gene Expression, Activity Assay, Methylation, MANN-WHITNEY