Pierce ecl plus chemiluminescent substrate

The protein concentration was verified by the method of Lowry33 (link). Cells and kidney tissues were lysed
with a 200-µL RIPA lysis buffer per plate (100 mm²). The lysates were
centrifuged at 12,000 g for 5 min at 4ºC, and the supernatants
were stored at −80ºC. Proteins (30 µg) were separated by 10% polyacrylamide gel
electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes
using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, CA, USA).
Nonspecific binding sites were blocked with 5% albumin (v/v) in TBS buffer. The
immunoblots were incubated overnight at 4ºC with renin (1:500, Santa Cruz, TX,
USA), angiotensin I (1:500, Santa Cruz, TX, USA), angiotensin II (1:500, Santa
Cruz, TX, USA), or GAPDH (1:500, Abcam, MA, USA) primary antibodies. After
washing three times with TBS-T, the membranes were incubated for 1 h at 4ºC in
HRP-conjugated secondary antibodies (1:100,000; Santa Cruz TX, USA).
Immunoreactive protein bands were visualized using Pierce ECL Plus
Chemiluminescent substrate (Thermo Fisher, USA). Images were obtained and
analyzed with an Alliance 7 Chemiluminescence documentation system (UVITEC,
Cambridge, UK). The immunoblot band intensities were quantified using Image J
software and reported as the renin/GAPDH, Angiotensin I/GAPDH, and Angiotensin
II/GAPDH ratio.

Total proteins were extracted from cellular pellets using protein extraction buffer (Triton X-100 1%, NaCl 150 mM, Tris 25 mM, pH 7.6) with Complete mini protease inhibitors (Roche). Thirty micrograms of protein were run on Nupage-NOVEX Bis-Tris 4–12% gels using a MOPS buffer. After electrophoresis proteins were transferred to a PVDF membrane (Invitrogen), and the membranes were then blocked for 1 h at room temperature with 5% non-fat milk dissolved on TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5). Primary antibodies Yap1 and GAPDH (Cell Signaling Technology) were added at a concentration of 1:1000 and were incubated overnight with the membrane at 4°C. HRP-linked anti-rabbit secondary antibodies (Cell Signaling Technology) were added at a concentration of 1:10,000 and incubated with membrane at room temperature for 1 hr. The proteins were visualized using a Pierce ECL Plus chemiluminescent substrate (Thermo Scientific).

For detection of autophagy biomarkers in vivo, tissues were snap frozen, and then mechanically disrupted before cell lysis. For ATG7 detection, cell lines were subjected to standard lysis procedures. In both cases, 50 µg proteins were separated on NuPAGE® Novex® Bis-Tris 4–12% pre-cast gels (Invitrogen) and electrotransferred to polyvinyldifluoride (PVDF) membranes (Millipore Sigma). Membranes were blocked with 0.05% Tween 20 (v/v in TBS) supplemented with 5% non-fat powdered milk for 1 h and incubated overnight with primary antibody specific for MAP1LC3B (1:1000, #2775 from Cell Signaling Technology), SQSTM1 (1:1000, #5114 from Cell Signaling Technology), ATG7 (1:1000, clone D12B11, #8558 from Cell Signaling Technology; or 1:3000, clone ATG7-13, #SAB4200304 from Millipore Sigma), or ACTB (1:1000, clone 13E5, #4970 from Cell Signaling Technology; or 1:2000, clone 8H10D10, #3700 from Cell Signaling Technology), at 4 °C. Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-mouse (#NA931, from GE Healthcare Life Sciences, 1:5000) or anti-rabbit (#NA934, from GE Healthcare Life Sciences, 1:5000) secondary antibodies and revealed with the Pierce™ ECL Plus chemiluminescent substrate (#32132, Thermo Fisher) on a C600 Gel Doc & Western Imaging System operated by cSeries Capture v. 1.6.8.1110 (Azure Biosystems).