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. 2020 Feb 18;9(2):472. doi: 10.3390/cells9020472

Figure 2.

Figure 2

Human ENSA and ARPP19 are substrates of both PKA and PKG. (a) The molecular weight of endogenous ENSA in human platelets and HEK293 cells is similar and is not affected by different treatments (thrombin (thr), iloprost (ilo) or sodium nitroprusside (SNP)). (b) Recombinant HisENSA wildtype and phosphosite mutants S109A and S109D were phosphorylated in the presence of the C-subunit of PKA or of PKGIβ (with 5 µM cGMP for PKG) and 1 mM ATP. Samples were taken in Laemmli buffer before (0 min) and after 0.5, 2 and 10 min of ATP addition and analysed by immunoblotting. HisENSA S109A and S109D mutants were not phosphorylated confirming S109 is the phosphorylated amino acid. (c) Recombinant GST-ARPP19 was phosphorylated in the presence of PKA C-subunit or PKGIβ (and 5 µM cGMP) and 1 mM ATP. Samples were taken in Laemmli buffer before (0 min) and after 5, 10 and 20 min of ATP addition. The western blots showing HisENSA and GST-ARPP19 phosphorylation are representative for n = 3 independent experiments.