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Figure S5. RNAi-mediated knockdown of TM5b promotes AQP2 trafficking. (A) Agarose gel analysis of RT-qPCR products of TM5b and GAPDH in MDCK cells transfected with siRNA against TM5b, a scrambled control siRNA, cells treated with cationic lipids alone (No siRNA), and untreated cells (Control). (B and C) MDCK cells were transfected with cDNA encoding GFP-AQP2 (green) and with either TM5b siRNA (C) or scrambled control siRNA (B), which was indicated by fluorescein-labeled dsRNA (red). The cells were pretreated with a PKA inhibitor H89 and then stimulated with forskolin. Bars, 10 µm. (D) Apical cell surface biotinylation assay using H89-pretreated cells corresponding to B and C. Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. The band density was normalized to the cells without siRNA transfection (No siRNA) and without forskolin stimulation. Data represent the mean and SE from three independent experiments. The single asterisk indicates a significant difference compared with the cells without siRNA and forskolin treatment (P < 0.05). The double asterisk indicates a significant difference compared with the control cells with forskolin stimulation (P < 0.05). (E) Molecular integrity of GFP-AQP2. 10 µg of protein of lysates of MDCK cells transfected with GFP-AQP2, AQP2, or GFP was analyzed by Western blot using antibodies for AQP2 or GFP (Clontech Laboratories, Inc.). In preparation of GFP-AQP2–transfected cells, a single band was observed at ∼58 kD corresponding to GFP-AQP2 in the immunoblotting for both AQP2 and GFP. (F) Apical cell surface biotinylation assay using the cells expressing WT-AQP2, S256A-AQP2, and S256D-AQP2. The cells were transfected with TM5b siRNA or scrambled control siRNA 48 h after transfection, and the cells were left untreated, stimulated with forskolin, incubated with mβCD, or stimulated with forskolin after preincubation with mβCD. The band density was normalized to WT-AQP2–expressing cells without siRNA and drug treatment. Data represent the mean and SE from three independent experiments. The single asterisk indicates a significant difference compared with WT-AQP2 expressing cells without siRNA and drug treatment (P < 0.05). The double asterisk indicates a significant difference compared with S256A-AQP2 expressing cells without siRNA and drug treatment (P < 0.05).