Fig. 1.

Phospho-antibody is specific for phosphorylation at urea transporter (UT)-A1 at Ser⁴⁹⁹ (S499). A: mIMCD3 cells transfected with UT-A1 (mIMCD3-UT-A1) were treated without (−) forskolin (FSK; vehicle) or with (+) FSK (10 μM) for 20 min at 37°C. After treatment, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Shown is a representative Western blot that was probed with UT-A1 antibody (top) or the generated phospho-specific antibody for S499 [phosphorylated (p)UT-A1/S499; bottom]. B: mIMCD3 cells were transfected with UT-A1 containing a site-directed mutation at S499 to alter this residue to an alanine (mIMCD3-UT-A1ΔS499A). Cells were then treated with FSK in a similar manner to mIMCD3-UT-A1. Western blot analysis confirmed that UT-A1ΔS499A expression was identified by the UT-A1 antibody at the expected molecular weight (top) and that pUT-A1/S499 recognized phosphorylation at S499 only (bottom). n = 4.