Western blotting

Cells were rinsed once with PBS, then lysed in sodium dodecyl sulfate lysis buffer with protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Roche) and gently scraped from hydrogels or tissue culture plates, and collected on ice. Cell suspensions were sheared by passing through a 27 guage needle, and centrifuged for 10 min at 10,000 g. Supernatants were collected and protein was quantified using bicinchoninic acid assays (Pierce). Equal amounts of protein were separated on 4–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. Samples were blocked in 5% milk and probed with primary antibody overnight at 4°C. Membranes were washed 3 × in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated in goat antirabbit (#W401B, Promega) or goat antimouse horseradish peroxidase (#7076P2; Cell Signaling) secondary antibodies (1:2000) for 1 h at room temperature. Membranes were washed 3 × in TBST and developed with Immobilon chemiluminescence substrate (Millipore) and visualized on a ChemiDoc MP Imaging System (Bio-Rad) or by exposure to X-ray film. Antibodies used were AQP1 (#AB2219) 1:1000 (Millipore), Claudin 2 (#12H12) 1:1000 (Invitrogen), GAPDH (#14C10) 1:5000, phosphorylated Smad2 (#3101) 1:1000 (Cell Signaling), and NHE3 (#sc-16103), 1:1000, (Santa Cruz).