Fig. 4.

MK2 levels are regulated by its interaction with p38α. (A and B) Lysates from CAFs (A) and the indicated mouse tissues (B) were immunoprecipitated with MK2-Trap_A (MK2) or agarose beads (Beads) and then were analyzed by immunoblotting. (C) CAFs were treated with UV-C 30 J/m² (UV30), and the cell lysates were immunoprecipitated with MK2-Trap_A (IP MK2) and then analyzed by immunoblotting. Band intensities were analyzed by ImageJ, and the p38α/MK2 ratios are indicated. (D) CAFs were treated with UV30 for the indicated times, and lysates were separated on 20 to 28% sucrose gradients. Collected fractions were analyzed by immunoblotting. PSMD11 was used as a marker for high-molecular-weight complexes. Band intensities were analyzed by ImageJ. The histograms show the p38α and MK2 levels in fractions 1 and 2, which are normalized to the total p38α and MK2 amounts in untreated cells (NT). Data represents mean ± SEM of two independent experiments. (E) CAFs were pretreated with the p38 inhibitor BIRB796 (p38 inh, 10 μΜ), to preserve the p38α–MK2 complex, or the vehicle dimethyl sulfoxide (DMSO) for 2 h and then incubated with CHX (50 μg/mL), which induces p38α activation and complex separation. At the indicated times, lysates were analyzed by immunoblotting. The estimated MK2 half-lives are indicated (t 1/2). Data represents mean ± SEM of three independent experiments. (F) Lysates from WT and p38α KO CAFs were separated on 20 to 28% sucrose gradients. Collected fractions were analyzed by immunoblotting. The asterisk in the blot indicates a nonspecific band. The 26S proteasome component PSMD11 was used as a marker for high-molecular-weight complexes. Band intensities were analyzed by ImageJ software. The histogram shows the quantifications of the MK2 levels in fractions 1 and 2, which are normalized to the total MK2 amount in WT cells. Data represents mean ± SEM of three independent experiments. Significant differences refer to fraction 2. (G) WT and p38α KO CAFs expressing Myc-p38α WT or the mutants K53M (KM) and Thr180A/Y182F (TY), or a GFP-expressing vector as control, were either left untreated or treated with UV30 for 1 h. Cell lysates were analyzed by immunoblotting. Band intensities were analyzed by ImageJ, and the MK2/Tubulin ratios are represented in the histogram. Data represents mean ± SEM of three independent experiments. When two MK2 bands were detected, the lower one (shorter isoform) was considered for quantification. **P < 0.01, ***P < 0.001.