Fig 1: Analysis of Protein Expression, Phosphorylation, and Cytokine Secretion in hiPSC-VSMCsWestern immunoblot (IB) analysis of PKG ic and PKG V219I hiPSC-VSMCs. Each line represents hiPSC-VSMC protein from 1 independent differentiation run (n = 4 independent runs per genotype). Immunoblot analysis was performed for (A) the VSMC-marker proteins MHY11, vinculin (VIN), and SMA. GAPDH was used as a loading control. (B) Additionally, protein expression was analyzed for COL4A1, COL1A1, CCN1, or CCN2, and (C) the cytoskeletal phosphorylation target of PKG-I, the vasodilator-stimulated phosphoprotein (VASP), as well as Ser239 phosphorylated VASP. GAPDH was used as a loading control. Western IB analysis was also performed to assess posttranslational modification of tubulin by tyrosination (Tyr-TUB) or detyrosination (deTyr-TUB). GAPDH was used as a loading control. Quantification of protein expression levels were normalized to GAPDH that was run on the same membrane. Cytokine production in hiPSC-VSMCs was performed in (D) cell lysates or (E) medium supernatant from PKG ic and PKG V219I hiPSC-VSMCs from day 22 of the differentiation. Pixel densities were quantified and normalized to the reference spots on each membrane. Data are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 by Student’s t-test. Figure 5 was part of Ms Schweizer’s master thesis. PAI-1 = plasminogen activator inhibitor 1; MCP-1 = monocyte chemoattractant peptide 1; other abbreviations as in Figures 1 and 5.