FIG. 4.

In vivo phosphorylation studies. (A) NDPK-A precipitations (IP) from 10 μg of liver cytosolic protein from wild-type littermate control (Wt), AMPK α1-null (α1 −/−), or AMPK α2-null (α2 −/−) mice run on 4 to 12% SDS-PAGE gels and probed with either a 1/1,000 dilution of anti-phosphoserine antibody or anti-NDPK-A antibody, as labeled. (B) HepG2 human-derived liver cell line transfected with various NDPK-A constructs: wild type, S122A, S144A, S120A, or empty vector control (mock). Transfected cells were either left untreated or incubated with 2 mM phenformin or 5 μg/ml oligomycin for 60 min. Ten micrograms of the cytosolic fraction from each construct was precipitated using an NDPK-A antibody as indicated, run on 4 to 12% SDS-PAGE gels, blotted, and probed using either a 1/1,000 dilution of phosphoserine or NDPK-A or ACC1 antibodies, as labeled. (C) Densitometry (histogram) data from four independent experiments were expressed in arbitrary units (AU) as a ratio of the phosphoserine amount over total protein amount ± standard error of the mean. (D) In the upper left panel, 10 μg of liver cytosolic protein from either wild-type (Wt), NDPK-A-null (A −/−), wild-type littermate control (Ct), AMPK α1-null (α1 −/−), or AMPK α2-null (α2 −/−) mice was run on 4 to 12% SDS-PAGE gels and probed with a 1/1,500 dilution of either anti-ACC1 antibody (leftmost blot) or anti-phospho-ACC1 antibody (center and right blots). In the lower left panel, 10 μg of the cytosolic fraction from mouse skeletal muscle from either wild-type (Wt), NDPK-A-null (A −/−), wild-type littermate control (Ct), AMPK α1-null (α1 −/−), or AMPK α2-null (α2 −/−) mice was run on an SDS-PAGE gel and probed with either a 1/1,500 dilution of anti-HSL antibody or a 1/2,000 dilution of phospho-HSL antibody. In each case, loading controls (LC) are included. Densitometry (D, right histograms) data from four independent experiments were calculated as described above for panel C.