Gene silencing with siRNA was performed as described previously (14 (link), 15 (link)). In brief, to deplete AVPR1A from the cell surface, cells were incubated in Nunc six-well plates (Thermo Fisher Scientific) for 3 d in Accell transfection media (GE Dharmacon) with AVPR1A or NT (negative control) siRNA (GE Dharmacon) at a concentration of 1 μM. On day 3, cells were centrifuged at 300g for 5 min and resuspended in RPMI 1640 (Sigma-Aldrich) supplemented with 10% HyClone FBS from Cytiva, 100 μg/ml penicillin from Invitrogen (Waltham, MA), and 100 μg/ml streptomycin (Invitrogen). Cells were used on day 4 for experimentation.
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