Western Blot Analysis of Urinary Exosomes

Urinary exosomal proteins were separated by SDS-PAGE (each sample was loaded in a lane with the same amount of creatinine) [22 (link),31 (link)] and then transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk in 0.05% Tween-Tris-buffered saline (TTBS), the membrane was incubated with 1.5% skim milk in TTBS including a primary antibody (anti-AQP2, -TSG101, or –ALIX antibody) at 30 °C for 1 h. The membrane was then incubated with 1.5% skim milk in TTBS including a secondary antibody at 30 °C for 45 min. Bands were visualized using a Super Signal chemiluminescence detection system (Thermo Fisher Scientific Inc., Waltham, MA, USA) and quantified using the ImageQuant TL software (GE Healthcare, Uppsala, Sweden). Corresponding control samples from animals treated with vehicle were always loaded in each gel for normalized quantification.