Skip to main content
. 2018 Mar 30;32(9):4941–4954. doi: 10.1096/fj.201701455RR

Figure 1.

Figure 1

Inducible fibroblast-specific deletion of p38α in mouse heart. A) Schematic diagram of the deletion strategy, combining Col1a2-Cre-ER(T) mice with floxed Mapk14 mice. Deletion of exons 2–3 occurs after tamoxifen injection, which activates Cre-ER(T). Red arrowheads denote position of genotyping primers X, Y, and Z (see Materials and Methods). B) Genotyping PCR showing effective exon 2/3 deletion in ear notch samples from tamoxifen-injected, experimental Cre-positive Mapk14fl/fl mice (E1–2) compared with control Cre-negative Mapk14fl/fl mice (C1–3). Top: Cre primers (Cre = 408 bp). Bottom: Mapk14 exon 2/3 floxed/deletion primers. Deletion (Z + Y) = 411 bp; floxed (X + Y) = 188 bp; and M = 100-bp ladder. C) Real-time RT-PCR analysis of Mapk14 mRNA levels in primary cultures of cardiac fibroblasts from control (Ctrl) Cre-negative Mapk14fl/fl mice (n = 6) and Cre-positive Mapk14fl/fl KO mice (n = 8) after tamoxifen injection. *P < 0.05. D) Immunoblot analysis of p38α protein in primary cultures of cardiac fibroblasts from control Cre-positive Mapk14wt/wt (C), heterozygous Cre-positive Mapk14wt/fl (Het), and experimental Cre-positive Mapk14fl/fl mice (E1–4) after tamoxifen injection. β-actin loading control. E) Densitometric analysis of p38α protein expression relative to β-actin in control, heterozygous (Het), and experimental (KO) cells. *P < 0.05. F) Characterization of nonmyocyte, isolated cell fractions from collagenase-digested mouse hearts (n = 7). Fr1, endothelial cells and leukocytes; Fr2, cardiac fibroblasts. Bar charts show quantitative RT-PCR data for mRNA levels of cell type–specific marker genes. Cardiomyocyte marker, Myh6; endothelial marker, Pecam1; fibroblast markers, Ddr2, Pdgfra, Col1a1, and Col1a2. All data normalized to Gapdh mRNA levels and expressed relative to whole heart. **P < 0.01, ***P < 0.001. G) Real-time RT-PCR analysis of Mapk14 mRNA levels in isolated cell fractions from hearts of control Cre-negative Mapk14fl/fl mice (n = 3) and experimental Cre-positive Mapk14fl/fl KO mice (n = 4) after tamoxifen injection. NS, not significant. **P < 0.01.