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. 2014 Jun 4;9(6):e98946. doi: 10.1371/journal.pone.0098946

Figure 4. Effect of inhibition of protein Ser/Thr phosphatases on N-terminal cGKI phosphorylation in intact cells.

Figure 4

Wild-type MEFs were incubated at 37°C under control conditions (1% DMSO in PBS for 15 min; Ctr), or for 15 min in the presence of 100 nM of the PP1/PP2A inhibitor, calyculin A (Cal A), or for 15 min in the presence of 100 nM calyculin A followed by 15 min with 1 mM 8-Br-cGMP (Cal A+8-cG) or 1 mM 8-Br-PET-cGMP (Cal A+PET-cG). Then the cells were lysed in denaturating buffer and cell lysates (10 µg) were analyzed by Western blotting with the indicated antibodies. GAPDH was used as loading control. The arrows indicate the positions expected for phospho-cGKI species as determined by co-loading of purified proteins on the same gel. Similar results were obtained in three independent experiments.