Fig. 5

The effect of PKC on phosphorylation of Nedd4–2 mutants. (A, top panel): Phosphorylation of single mutants of Nedd4–2. OAT1-expressing COS-7 cells were transfected with individual single mutants of Nedd4–2: Thr197Ala (T197A), Ser221Ala (S221A), Ser354Ala (S354A), and Ser420Ala (S420A). Transfected cells were incubated with 10 μM PMA for 1 h. Cells were lysed and Nedd4–2 mutants were immunoprecipitated (IP) with anti-FLAG antibody (epitope FLAG was tagged to all Nedd4–2). Normal mouse IgG was used as non-specific binding control (IgG). Then immunoblotting (IB) was performed with anti-phospho-Ser/Thr primary antibody and anti-rabbit secondary antibody. (A, bottom panel): The same immunoblot from the top panel was stripped and reprobed with anti-FLAG antibody to determine the total amount of Nedd4–2 pulled-down. (B): Densitometry plot of results from (A) as well as from other repeat experiments. The values are mean ± S.D. (n = 3). *p < 0.05. (C, top panel): Phosphorylation of quadruple mutant of Nedd4–2. OAT1-expressing COS-7 cells were transfected with either wild type (WT) or quadruple mutant (Quad) of Nedd4–2. Transfected cells were incubated with 10 μM PMA for 1 h. Cells were lysed and Nedd4–2 (wild type and mutant) was immunoprecipitated (IP) with anti-FLAG antibody (epitope FLAG was tagged to all Nedd4–2). Normal mouse IgG was used as non-specific binding control (IgG). Then immunoblotting (IB) was performed with anti-phospho-Ser/Thr primary antibody and anti-rabbit secondary antibody. (C, bottom panel): The same immunoblot from the top panel was stripped and reprobed with anti-FLAG antibody to determine the total amount of Nedd4–2 pulled-down. (D): Densitometry plot of results from (A) as well as from other repeat experiments. The values are mean ± S.D. (n = 3). *p < 0.05