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. 2023 Mar 28;324(5):L700–L711. doi: 10.1152/ajplung.00340.2022

Figure 4.

Figure 4.

MK2 associates with caspase-3 and MK2’s nuclear translocation is required for increased caspase-3 activation and resultant apoptosis. A: H23 cells were infected with Ad-WT-MK2, Ad-WT-MK2-BioID-C or Ad-WT-MK2-BioID-N (MK2 fused to biotin ligase on the c-terminus or n-terminus) and following incubation with biotin, cell lysates were precipitated with beads conjugated to streptavidin and then immunoblotted using antibodies directed against caspase-3. There is increased biotinylated caspase-3 with biotin ligase fused to MK2 on the c-terminus. B: H23 cells were infected with Ad-eGFP, Ad-WT-MK2, Ad-Mut-NLS-MK2 (mutated nuclear localization sequence, rendering cytosolic sequestration), Ad-Mut-NES-MK2 (mutated nuclear export sequence, limiting nuclear export) and 48 h afterwards nuclear and cytosolic fractions were separated for analyses. Representative immunoblot shows wild-type and mutant NES-MK2 constructs translocate to the nuclear compartment, whereas the mutant NLS-MK2 construct is sequestered in the cytosolic fraction and unable to translocate to the nucleus. C: H23 cells were infected with Ad-WT-MK2, Ad-Mut-NLS-MK2, Ad-Mut-NES-MK2 and 48 h afterwards cell lysates were immunoprecipitated with antibodies against MK2 and then immunoblotted using antibodies directed against caspase-3. As shown, caspase-3 is associated with MK2. There is no difference in association based on localization mutations. D: H23 cells were infected with Ad-eGFP, Ad-WT-MK2, Ad-Mut-NLS-MK2 and Ad-Mut-NES-MK2 and 48 h afterwards cell lysates were analyzed for caspase-3 activity. Cells infected with wild-type and mutant NES-MK2 constructs exhibited a significant increase in caspase-3/7 activity compared with H23 cells infected with Ad-eGFP or Ad-Mut-NLS-MK2. *P < 0.05 vs. Ad-WT-MK2 and Ad-Mut-NES-MK2 in post hoc testing using Tukey’s multiple comparisons test. n = 3–6 per group. E: H23 cells were infected with Ad-eGFP, Ad-WT-MK2, Ad-Mut-NLS-MK2 and Ad-Mut-NES-MK2 and 48 h afterwards cells were analyzed for cell death using flow cytometry. Cells infected with wild-type and mutant NES-MK2 constructs exhibited a significant increase in cell death compared with H23 cells infected with Ad-eGFP or Ad-Mut-NLS-MK2. *P < 0.05 vs. Ad-WT-MK2 and Ad-Mut-NES-MK2 in post hoc testing using Tukey’s multiple comparisons test. n = 9 per group. MK2, mitogen-activated protein kinase-activated protein kinase 2; NLS, nuclear localization sequence; NES, nuclear export sequence.