Fig. 3

The effect of PKC on OAT1 ubiquitination. (A, top panel): OAT1 ubiquitination. OAT1-expressing COS-7 cells were transfected with wild type Nedd4–2. Transfected cells were incubated with 10 μM PMA for 1 h, with or without 2 μM staurosporine. Cells were lysed and OAT1 was immunoprecipitated (IP) with anti-myc antibody (epitope myc was tagged to OAT1). Normal mouse IgG was used as non-specific binding control (IgG). Then immunoblotting (IB) was performed with anti-ubiquitin primary antibody and anti-mouse secondary antibody. (A, bottom panel): The same immunoblot from the top panel was stripped and reprobed with anti-myc antibody to determine the total amount of OAT1 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