Journal: Journal of the American Society of Nephrology : JASN

Article Title: Akt Substrate of 160 kD Regulates Na + ,K + -ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

doi: 10.1681/ASN.2013101040

Figure Lengend Snippet: Energy depletion leads to AS160 dephosphorylation at S588. (A) The apparent molecular weight of AS160 decreases as a result of its dephosphorylation. MDCK cells are subjected (AA/DG) or not (−) to energy depletion for 1 hour. Total cell lysates are incubated in the presence (+) or absence (−) of 10 units of CIP for 30 minutes at 37°C. Lysates are blotted with anti-AS160 antibody to visualize the shift of the apparent molecular weight of AS160. Blots are also probed with anti–phospho-AMPK and p-AS160 S588 antibodies to document the efficacy of both the ATP depletion and the CIP treatments. Anti–β-actin is also used to assess the total amount of protein loaded. (B) Total MDCK cells lysates are blotted with rabbit anti-AS160 to detect the endogenous AS160 and with phospho-specific antibodies directed against the AS160 phosphorylation sites S588 and T642. Lysates are also probed with antibodies directed against p-AMPK. Blots are probed with anti–β-actin to assess the total amount of protein loaded. The data indicate that energy depletion is associated with a decrease in the apparent molecular weight of AS160 that is induced by the dephosphorylation of AS160 at S588. (C) Quantification of AS160 phosphorylation at S588 and T642 is based on four independent experiments. *P<0.01. Typical results of one of four experiments are shown. CIP, calf intestinal alkaline phosphatase; p-AMPK, phospho-AMPK.

Article Snippet: 45 Polyclonal phospho-specific antibodies recognizing AS160 phosphorylated at S588 and T642 were obtained from Symansis (New Zealand).

Techniques: De-Phosphorylation Assay, Molecular Weight, Incubation