Figure 4.

Longitudinal stretch regulates Ankrd2 through Akt dependent NF-κB signaling pathway. Diaphragms of WT mice were preincubated with or without 5 μM Akt inhibitor IV, 10 μM IKK inhibitor BAY 11-7082, 20 μM p50 inhibitor peptide, or 50 μM p65 inhibitor peptide for 30 min. After preincubation, the diaphragms were subjected to stretch longitudinally to the myofibers for 15 min or maintained under unstretched conditions. A) Cytoplasmic and nuclear lysates prepared from each diaphragm (85 μg/sample) were resolved by SDS-PAGE gel and immunoblotted with ether normal or phospho-specific p50 or p65 antibody. Gel pictures are representative of 3 independent experiments (n=3). B) Top panels: total RNA (1 μg) prepared from each diaphragm was subjected to cDNA synthesis, and equal amounts of cDNA from the above samples were subjected to real-time qPCR assays to estimate Ankrd2 and GAPDH (as a normalizer) mRNA levels. Level of Ankrd2 mRNA is presented relative to that of GAPDH mRNA level. Values represent the mean ± se of 3 independent experiments. *P < 0.05 vs. corresponding stretch sample. Bottom panels: cytoplasmic and nuclear lysates prepared from each diaphragm (85 μg/sample) were resolved by SDS-PAGE gel and immunoblotted with ether Ankrd2 or tubulin (loading control) antibody. Gel pictures are representative of 3 independent experiments (n=3).