FIGURE 4.
Heterologously expressed WT telethonin and endogenous telethonin are constitutively phosphorylated. A, Phos-tag SDS-PAGE and immunoblot (IB) analysis of ARVM infected with AdV:HA-WT-telethonin or AdV:HA-S157A/S161A-telethonin for 42 h and stimulated with vehicle, ET-1 (100 nm), or PE (10 μm, with 1 μm atenolol) for 10 min. Cell lysates were subjected to Phos-tag SDS-PAGE and immunoblot analysis using a monoclonal anti-HA antibody (top panel) or a monoclonal anti-telethonin antibody (bottom panel). The positions of internal standards resolved in the same gel are marked by thick black lines to the left of the immunoblot obtained with the anti-telethonin antibody, indicating the positions of dual-phosphorylated, mono-phosphorylated, and non-phosphorylated His6-tagged WT telethonin from top to bottom. Arrows on the right show the deduced identities of the indicated telethonin species. 2P-HA, dual-phosphorylated HA-tagged heterologous telethonin; 1P-HA, mono-phosphorylated HA-tagged heterologous telethonin; 0P-HA, non-phosphorylated HA-tagged heterologous telethonin; 2P, dual-phosphorylated endogenous telethonin; 0P, non-phosphorylated endogenous telethonin. B, the same samples were subjected to standard SDS-PAGE and immunoblot analysis using the same antibodies (n = 3).