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. 2016 Aug;55(2):275–287. doi: 10.1165/rcmb.2015-0185OC

Figure 1.

Figure 1.

Asymmetric dimethylarginine (ADMA) induces Akt1 phosphorylation in pulmonary arterial endothelial cells (PAECs). PAECs were exposed to ADMA (10 μM, 2 h) and whole-cell extracts (20 μg) subjected to immunoblot (IB) analysis using antibodies raised against pThreonine (T) 308Akt1 (pT308Akt1), pSerine (S) 473Akt1 (pS473Akt1), or phosphatase and tensin homolog deleted on chromosome 10 (pPTEN). Blots were then stripped and reprobed using antibodies raised against β-actin, Akt1, or PTEN. Representative images from duplicate blots are shown. ADMA significantly increased Akt1 phosphorylation at T308 (A) and S473 (B). PTEN activity was unchanged, as estimated by measuring pPTEN levels (C). Plasma membrane protein extracts (20 μg) were also subjected to IB analysis using an antibody raised against Akt1. Reprobing the plasma membrane IBs with an antibody raised against NaKATPase was used to normalize loading. Plasma membrane IBs were reprobed with antibodies raised against laminB1 or 58K to demonstrate no cross-contamination with nuclear or Golgi fractions, respectively. A separate gel was run using a PAEC whole-cell lysate (20 μg) to confirm that each antibody recognizes the ovine protein (D). ADMA does not change Akt1 levels in membrane extracts (D). Changes in plasma membrane and cytosolic Akt1 levels in response to ADMA were also evaluated by confocal microscopy. Representative images, including insets with additional higher magnifications, are shown (E). After converting every channel to a binary mask, signals were subtracted from each other to obtain cytosolic, membrane, and nuclear localized Akt1. There was a significant increase in Akt1 at the membrane and a significant decrease in the cytosol after ADMA treatment (E). The level of Akt1 at the plasma membrane combined with the Akt1 in the cytosol was unchanged, suggesting that total Akt1 levels were unchanged. Akt1total, total Akt1; Akt1cyt, cytosolic Akt1; Akt1mem, membrane Akt1; Akt1nuc, nuclear Akt1; DAPI, 4′,6-diamidino-2-phenylindole; neg. control, negative control. Scale bars: 10 μm. Values are means (±SEM); n = 3–7. *P < 0.05 versus untreated, **P < 0.01 versus untreated. ns, not significant.