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. 2019 Jul 1;30(14):1664–1675. doi: 10.1091/mbc.E18-07-0438

FIGURE 4:

FIGURE 4:

Sun2-/- hearts display increased integrin engagement and AKT/MAPK signaling. (A) Frozen P50 WT and Sun2-/- cardiac left ventricle tissue was sectioned and stained with antibodies against total β1-integrin (top panels) or against the ligand-bound, active β1-integrin (9EG7) conformational epitope (bottom panels). Images were pseudocolored to depict the relative fluorescence intensity of the β1-integrin signal, with lighter colors indicative of higher intensity and darker colors indicative of lower intensity. Note the increased intensity of active β1-integrin in the Sun2-/- tissue. Images are representative of results for 3 mice per genotype (also see Supplemental Figure 2, E–G). (B–E) Representative immunoblots of ventricular lysate from three WT and three Sun2-/- mice at P50 or five WT and four Sun2-/- mice at 13 mo. Ponceau staining of total protein reveals even loading of samples. Plots to the right represent quantitative analysis from additional mice, represented as the mean ± SD for more than three WT or Sun2-/- mice. Full blots, used for quantifications, are shown in Supplemental Figure 3. For quantification, data are shown as a ratio of Sun2-/- to WT for the phosphorylated protein and total protein, expressed as a ratio. (B) Lysates from P50 mice were subjected to SDS–PAGE and immunoblotting with antibodies against phosphorylated AKT (pAKT), AKT, phosphorylated S6 (pS6), or S6, revealing elevated levels of pAKT in Sun2-/- tissue. Data are represented as the mean ± SD for three WT or Sun2-/- mice. (C) Lysates from P50 mice were subjected to SDS–PAGE and immunoblotting with antibodies against pERK1/2 and ERK2, revealing elevated levels of pERK1/2 in Sun2-/- tissue. Data are represented as the mean ± SD for three WT or Sun2-/- mice. (D) Lysates from 13-mo-old mice were subjected to SDS–PAGE and immunoblotting with antibodies against phosphorylated S6 (pS6), S6, phosphorylated AKT (pAKT), or AKT, revealing elevated levels of pS6 but similar levels of pAKT in Sun2-/- tissue vs. WT tissue. Data are represented as the mean ± SD for five WT or four Sun2-/- mice. (E) Lysates from 13-mo-old mice were subjected to SDS–PAGE and immunoblotting with antibodies against pERK1/2 and ERK2, revealing similar levels of pERK1/2 in Sun2-/- and WT tissues. Data are represented as the mean ± SD for three WT or Sun2-/- mice.