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. 2022 Dec 20;119(6):1361–1376. doi: 10.1093/cvr/cvac191

Figure 4.

Figure 4

Identification of CARMEN-201 protein partners. (A) Quantification of REST, (B) NSUN6 and (C) RPA1 by Western blotting in a protein pulldown assay using biotinylated sense or antisense CARMEN-201 transcript in adult CPC lysates. Graphs show mean values ± SEM *P < 0.05 as compared with input; §P < 0.05 comparing sense and antisense probe (n = 3). (D–F) Quantification of CARMEN-201 enrichment after RNA immunoprecipitation using a control immunoglobulin G (IgG) or IgG directed against REST (anti-REST IgG), NSUN6 (anti-NSUN6 IgG) and RPA1 (anti-RPA1 IgG). Graphs show mean values ± SEM; *P < 0.05 as compared with control (n = 3). (G) Expression of REST, CARMEN-201 and SMC markers (MYH11; CNN1; TAGLN) in differentiating fetal CPCs either not transduced (None), transduced with a lentiviral vector encoding C-201 Exon 2 (C-201 Ex2), and treated with either a scrambled siRNA (Scr siRNA) or a siRNA directed against REST (Anti-REST siRNA). (H) Representative images and quantification of SMMHC-positive CNN1-positive SMCs and ACTN2-psoitive CMs in cultures of differentiating fetal CPCs transfected as in (G). Scale bars: 50 µm. Data represent mean values ± SEM; *P < 0.05 as compared with fetal CPCs in expansion; §P < 0.05 compared with indicated conditions (n = 3–6). ANOVA with post hoc Tukey. See also Supplementary material online, Figure S6.