Semiquantitative Immunoblotting of AQP2 and pAQP2

The cell lysates were obtained in 1X Laemmli buffer (10 mM Tris-HCl, pH 6.8, 1.5% SDS), including protease and phosphatase inhibitor cocktail (Halt^TM Protease and Phosphatase Inhibitor Cocktail 100X, 78440, Thermo Fisher Scientific, Waltham, MA, USA). They were placed in the QIAshredder column (79656, QIAGEN, Hilden, Germany) and centrifuged at 10,000× g for 2 min at room temperature. The total protein concentration was measured using the BCA assay kit (Pierce BCA protein assay reagent kit; 23227, Thermo Fisher Scientific, Waltham, MA, USA). Semiquantitative immunoblotting was performed, as previously described [21 (link),22 (link)]. Primary antibodies used were anti-AQP2 (1:1000, AB3274, Merk Millipore, Burlington, MA, USA), anti-phosphorylated-AQP2 at serine 256 (1:1000, K0307AP) [30 (link)], and anti-β-actin (1:400,000, A1978, Sigma, St. Louis, MO, USA). Immunoblots were visualized by horseradish peroxidase-conjugated secondary antibodies (P447, P448, DAKO, Glostrup, Denmark). Densitometric values were corrected by the densitometry value of β-actin and band density was quantitated by ImageJ (NIH).