Dry skim milk

Mice kidneys were homogenized and incubated with radio-immunoprecipitation assay (RIPA) lysis buffer (Cell Signaling, USA) containing proteases and phosphatases inhibitors (Sigma, Israel) in ratio of 1 : 100 and 1 : 1000, respectively. Kidney's lysate was centrifuged, and the supernatant containing total proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% dry skim milk (Bio-Rad, CA, USA) in Tris-buffered saline with Tween 20 at room temperature and washed and incubated with primary antibodies (Anti-APG5L/ATG5 ab109490 [EPR4797] 1 : 1000; Anti-LC3-II ab48394 1 : 1000; Anti-GAPDH ab181602 1 : 10000) at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated to secondary antibody at room temperature. Bands were visualized using clarity enhanced chemiluminescence kit (Bio-Rad, CA, USA) and were analyzed using Bio-Rad image Lab software, are data presented in arbitrary units (AU).

Renal tissues were homogenized with lysis buffer containing protease (#P8340, Sigma, Israel) and phosphatase inhibitors (#04906837-001, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland). Kidney lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which proteins were transferred to nitrocellulose or polyvinylidene difluoride (PDVF) membranes. The membranes were then blocked with 5% dry skim milk (Bio-Rad, USA) in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h, washed three times with TBST, and incubated with primary antibodies in 5% dry milk in TBST, at 4°C, overnight. The membranes were washed with TBST and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody at room temperature, for 35 minutes, followed by TBST washes. Bands were visualized using Clarity Enhanced Chemiluminescence (ECL) Kit (Bio-Rad, USA), and band intensities were analyzed using Image Lab software (Bio-Rad, USA).