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. 2018 Sep 7;9(9):450. doi: 10.3390/genes9090450

Figure 3.

Figure 3

Activity of full-length ppAkt1 and commercially available active Akt1. (A) Kinase activity assays over a 30 min time course show substantially reduced activity of commercial Akt1 (lot 1, green squares) compared to full-length ppAkt1 (blue diamonds) produced in E. coli. Commercial Akt1 lot 2 (red circles) showed highly variable but similar activity to full-length ppAkt1. (B) Tryptic peptides from commercial Akt1 and ppAkt1 were analyzed by parallel-reaction monitoring mass spectrometry (PRM-MS). The purchased active Akt1 (lot 1) showed a low-intensity peak for phosphorylation at Thr308 (green peak at a retention time of ~40 min) and high-intensity peak for non-phosphorylated Thr308 (cyan peak, retention time of ~37 min). (C) PRM-MS analysis of ppAkt1T308,S473 showed a high-intensity peak for phosphorylation at Thr308 (green peak at a retention time of 40 min) and the non-phosphorylated Thr308 was undetectable.