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. 2023 Jul 4;26(8):107283. doi: 10.1016/j.isci.2023.107283

Figure 4.

Figure 4

The aging-associated deleterious activation of ERK can be rescued by Kindlin-2 overexpression in the old cells

(A) Representative Western blot showing the difference in phosphorylated ERK1/2 (P-ERK) versus total ERK1/2 protein levels in the lysate from old and young male cardiac fibroblasts. Densitometry was presented on an individual-plotted diagram (N = 5–6 per group).

(B) Representative Western blot depicting changes in F- and G-actin fraction enrichment in the cells cultured with 5 μM Temuterkib (ERK1/2 specific inhibitor, also known as LY3214996, “ERK-LY”) or DMSO (control, “-”) for 3 h. The F-to-G-actin ratio was calculated (lower panel) (N = 3–5 per group).

(C–E). Representative Western blot showing the change in α-SMA and Kindlin-2 in the lysate of old male cells treated for 72 h with 5 μM LY3214996 (ERK-LY, “+”) or DMSO (control, “-“). For each protein, the normalized densitometry of each individual value is plotted in a diagram (lower panel) (N = 4–6 per group, “ERKinh” depicts cells treated with 5μM Temuterkib). Staining for F-actin (red)/α-SMA (green) (D.) and Kindlin-2 (green)/F-actin (red) (E.) on cells treated with 5 μM Temuterkib (ERK inhibitor) or DMSO (control) for 72 h. In E., arrows indicate the location of Kindlin-2 at the plasma membrane, and “N” its perinuclear location. Scale bar: 20 μm. Pictures in enlarged frames (yellow square) depicted the changes in Kindlin-2 localization between control and Temuterkib-treated cells. White arrows indicate focal adhesion localization, while “N” depicts nuclear/perinuclear localization of Kindlin-2 (Scale bar: 5 μm).

(F) Representative Western blot depicting the level of phosphorylated ERK1/2 (P-ERK) and ERK1/2 in the lysate of pre-selected clones overexpressing Kindlin-2 (“K-OE + ”) or a control plasmid “K-OE -“). The P-ERK to ERK ratio was calculated and shown in an individual-plotted diagram (N = 6 per group). M denotes a molecular weight marker; the corresponding molecular weight is indicated on the right side of the blot.All the data are shown as an averaged mean ± SEM. Statistical significance was challenged by an unpaired t-test with Welch’s correction. p value <0.05 is considered statistically significant.