Af1513

Protein samples were supplemented 5 × concentrated SDS-PAGE sample buffer containing β-mercaptoethanol and boiled at 100 °C for 5 min. Then, they were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (GE). Membranes were blocked with 5% nonfat milk for 1 h, followed by incubation with the primary antibodies: anti-PRSS55, anti-GAPDH (2118S, Cell signaling technology), anti-Lamin A/C (2032T, Cell signaling technology), anti-NaKATPase (3010S, Cell signaling technology), anti-uPAR (ab103791, Abcam), anti-ACTB (sc-47778, Santa Cruz), anti-ADAM3 (sc-365288, Santa Cruz), anti-ADAM2 (MAB19292, Millipore), anti-CLGN (ab171971, Abcam), anti-PDILT (ab116182, Abcam), anti-PRSS37 (HPA020541, Sigma-Aldrich), anti-TEX101 (ab69522, Abcam), anti-tACE (AF1513, R&D Systems), anti-ATP5A1 (14676-1-AP, Proteintech) and anti-GFP (G1544, Sigma). To visualize specific protein bands, secondary antibodies conjugated with IRdye800CW (LI-COR, Lincoln, USA) were used and membranes were scanned by Odyssey Infrared Imager (LI-COR). ACTB, GAPDH or ATP5A1 were used as protein loading controls.

Immunofluorescence against aldose reductase and ACE was performed on 10% goat serum in phosphate-buffered saline blocked kidney slices further incubated overnight with primary antibodies (AR antibody, kind gift from Dr Mark Petrash and ACE antibody (AF1513, R&D)). The following day, slides are rinsed and incubated with Alexa Fluor-conjugated secondary antibody (Molecular Probes) against the specific IgG of the primary antibody. For nucleus identification, DAPI staining is used in combination with a fluorescence fading retardant (Vector Laboratories, Burmingdale, CA) before imaging by confocal microscopy. Immunostained preparations were imaged and analysed using a laser-scanning confocal microscope (LSM510, Carl Zeiss, Thornwood, NY) with a × 40 water immersion objective and the corresponding postacquisition software. Immunofluorescence analysis was performed from sections from three animals and by evaluation of >10 random fields each66 (link).
Renal oxidative stress by dihydroethidium staining was determined following the manufacturer's protocol (D1168, Thermo).