Figure 6.

β₁Pix regulates AMPK-dependent Nedd4-2–14-3-3 association and Nedd4-2 cellular stability in mCCD_cl1 cells. A, representative immunoblot (Input) and co-IP results of β₁Pix, Nedd4-2, and 14-3-3 proteins in total lysates from polarized mCCD_cl1 cells with or without β₁Pix knockdown after 24-h treatment with or without AMPK activators (AA). Ab, antibody. B, summary graph of the ratio of pAMPK-α (Thr-172) to total AMPK-α and β₁Pix protein expression levels. IB, immunoblot. C, summary graph of the co-immunoprecipitated β₁Pix (left panel) or Nedd4-2 (right panel) with 14-3-3 proteins under the indicated conditions. D, immunoblot analysis of Nedd4-2 protein expression in cells with or without β₁Pix knockdown and with or without AA treatment. E, RT-PCR analysis of Nedd4-2 mRNA in cells with or without β₁Pix knockdown and with or without AA treatment. F, phosphorylation of Nedd4-2 (Ser-328) in cells with or without β₁Pix knockdown and with or without AA treatment. G, Nedd4-2 cellular stability in control versus β₁Pix knockdown cells. Cells were treated with cycloheximide (100 μg/ml) and harvested at different time points (*, p = 0.008 for comparison of control and β₁Pix knockdown at the 2-h time point). Immunoblot assays were performed to detect changes in cellular Nedd4-2 protein abundance. Data are mean ± S.E. (p values for the indicated comparisons with significant differences are shown).