Sc 20631

The protein samples were gathered from the cells with an RIPA buffer (R0278, Merck/Millipore Sigma) or the fractionation step. Subsequently, the protein concentration was determined using a protein assay dye (5000006, BIO-RAD, Hercules, CA, USA) and access to an equal amount of protein, then separated by electrophoresis on SDS-PAGE. After the proteins were transferred on polyvinylidene difluoride (PVDF) membranes (1620177, BIO-RAD), the PVDF membrane was immersed in 5% skim milk at 1X TBST, blocking for 1 h at room temperature, and washed three times with 1X TBST for 5 min. Next, we incubated the PVDF membrane with the primary antibody at 4 °C overnight, washed it three times with 1X TBST for 5 min, and incubated it with the secondary antibody for 1 h at room temperature. It was washed three times with 1X TBST for 5 min after secondary antibody incubation, then we added the enhanced chemiluminescence (ECL) substrate kit (GERPN2134, Merck/Millipore Sigma) and detected the protein signal using an X-ray processor (MXP-101, KODAK, Rochester, NY, USA) on X-ray film (Super RX, FUJIFILM, Minato-ku, TKY, JPN). Primary and secondary antibodies were diluted with 5% skim milk in 1X TBST, and the detailed information isas follows: 1:2000 for uromodulin (sc-20631, Santa Cruz, Dallas, TX, USA) and 1:20,000 for rabbit HRP 2nd antibody (GTX213110-01, GeneTex, Irvine, CA, USA).

Following antibodies were used for immunostaining: anti-green fluorescent protein (GFP) (1:1000, MAB3580, Merck Millipore, Billerica, Massachusetts, USA), anti-Syntenin-1 (1:1000, ab133267, Abcam, Cambridge, UK), anti-Flotillin-1 (1:1000, 610820, BD Biosciences, Franklin Lakes, New Jersey, USA), anti-Ago2 (1:1000, ab32381, Abcam, Cambridge, UK), anti-Alix (1:1000, 2171 S, Cell Signaling Technology, Beverly, Massachusetts, USA), anti-CD81 (1:1000, SC-166029, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-TSG101 (1:1000, SC-7964, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-Tamm-Horsfall (1:1000, SC-20631,, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-ApoA-1 (1:1000, SC-376818, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-albumin (1:1000, an28405, Abcam, Cambridge, UK), anti-IgG (1:1000, ab181236, Abcam, Cambridge, UK), anti-PMP70 (1:250, P0497, Sigma-Aldrich, St. Louis, Missouri, USA), sheep anti-mouse horseradish peroxidase-linked antibody (1:3000, NA931V, GE Healthcare Life Sciences, Uppsala, Sweden), donkey anti-rabbit horseradish peroxidase-linked antibody (1:4000, NA934V, GE Healthcare Life Sciences, Uppsala, Sweden).

Immunohistochemistry was performed on 3 μm-thick paraffin-embedded tissue sections. Tissue was de-paraffinized, and 10 mM citric acid, pH 6, at 95 °C, was used for antigen retrieval. Sections were permeabilized with 0.1% triton/PBS. Antibodies against Notch3 (Abcam, Cambridge, UK, ab23426), F4-80 (AbD Serotec, Oxford, UK, MCA497R), MCM2 (Cell Signaling, Danvers, MA, USA, #4007), pax2 (Abcam, ab150391), PCNA (Merck, Darmstadt, Germany, #NA03), aquaporin 1 (Alpha diagnostic AQ11-A), aquaporin 2 (Abcam, ab15116), CD13 (Abcam, ab 108310) and Tamm–Horsfall protein (Santa Cruz, Dallas, TX, USA, sc-20631) were used. Immunofluorescence for LC-3 (Novus Bio, Littleton, CO, USA, 100-2220) was performed on 3 μm-thick paraffin-embedded tissue in a similar manner. Alexa fluor (Invitrogen) secondary antibody was used for detection. Images were obtained with an OlympusIX83 photonic microscope at ×200 magnification.