protein kinase pp38mapk  (Cell Signaling Technology Inc)

Journal: JACC: Basic to Translational Science

Article Title: A New Ex Vivo Model to Study Cardiac Fibrosis in Whole Mouse Hearts

doi: 10.1016/j.jacbts.2024.04.007

Figure Lengend Snippet: Molecular Regulation of Fibrosis in Ex Vivo Cultured Hearts Representative pictures of the right ventricle and the right atrium of hearts cultured for 7 days under standard conditions (−), in the presence of SB431542 (SB, SB1, SB2) or transforming growth factor-beta (TGF-β) for (A) phospho-SMAD2/3 (pSMAD2/3) and phospho-p38 mitogen-activated protein kinase (pp38MAPK), (B) α-SMA and vimentin, (C) PECAM-1 and cTnI, (D) collagen I and collagen III, and (E) Alcian blue, periostin, versican B, and fibronectin. (F) Graphs depicting the percentage of hearts with α-SMA expression in the subepicardium of the ventricle and atrium after culture for 7 days under standard conditions (−), in the presence of SB431542 (SB) or TGF-β. (G and H) Graphs depicting the relative α-SMA-positive area (G) and the relative vimentin-positive area (H) in the ventricular or atrial myocardium of hearts cultured for 7 days under standard conditions (−), in the presence of SB431542 (SB) or TGF-β. DAPI was used as counterstain in all fluorescent pictures. Data are presented as means ± SEM. To evaluate significant differences, 1-way analysis of variance followed by Tukey’s multiple comparisons test was performed for graphs (G and H). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed for graph (G [atrium]). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Scale bar: 20 μm. Abbreviations as in Figure 1 .

Article Snippet: After blocking with 1% BSA in 0.1% Tween-PBS, sections were incubated overnight with the primary antibodies directed against platelet endothelial cell adhesion molecule (PECAM-1) (R&D, AF3628; 1:1,000), proliferating cell nuclear antigen (PCNA) (Sigma, P8825; 1:1,000), smooth muscle actin-alpha (α-SMA, Sigma; A2547; 1:20,000), collagen I (SouthernBiotech, 1310-01; 1:200), GFP (Abcam, ab13970, 1:1,000), collagen III (SouthernBiotech, 1330-01; 1:200), phospho-p38 mitogen-activated protein kinase (pp38MAPK) (Cell Signaling, #4631; 1:100), periostin (Santa Cruz, SC398631; 1:100), versican B (Chemicon, AB1033; 1:100), fibronectin (Sigma, F3648; 1:200), cardiac troponin-I (cTnI) (HyTest, 4T21/2; 1:1,000), matrix metalloproteinase 2 (MMP2) (Invitrogen, #436000; 1:200), vimentin (Cell Signaling, #5741; 1:200), mannose receptor (CD206, Abcam; ab64693; 1:300), F4/80 (Invitrogen, 14-4801-82; 1:100), MAC3 (CD107b, BD Biosciences, #550292; 1:200), CD68 (Santa Cruz, SC20060; 1:100), and sarcomeric myofilaments (MF20, Developmental Studies Hybridoma Bank, University of Iowa) followed by incubation with Alexa-conjugated secondary antibodies (Molecular Probes).

Techniques: Ex Vivo, Cell Culture, Expressing