Figure 3.

PP1 interacts with NCC and dephosphorylates it. (A) FLAG IP pull down of NCC (left) and the catalytic subunit of PP1 (PPIc) (right). Unrelated anti-AQP1 antibody as well as no antibody were used as negative control. (B) In vitro dephosphorylation of biotinylated pT58-mNCC peptide by PP1. (C) Changes in NCC phosphorylation at T58 in MDCK type I cells with tetracycline-inducible FLAG-tagged NCC expression upon treatment with calyculin A (left panel) or specific PP2A inhibitor endothall (right panel). Graph represents the densitometric quantification of immunoblots from two independent experiments. *P<0.05 by one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Changes in NCC phosphorylation upon treatment of WT and I1-KO mouse kidney slices with calyculin A (20 nmol/L). Bar charts represent the densitometric quantification of pT53NCC/tNCC from six slices (in brackets) normalized to control vehicle of each genotype (two mice per group). Error bars represent the SEM. ****P<0.0001 assessed by two-way ANOVA followed by Tukey’s multiple comparison post-test.