Ecl reagent kit

The appropriate amount of renal cortex was placed into homogenate tubes with an equal proportion of RIPA lysis buffer (Thermo Fisher) mixed with a protease inhibitor cocktail (Roche) and homogenized using a tissue grinder. Protein lysates were then prepared for western blot analysis. Proteins were isolated by electrophoresis on 12.5% SDS–PAGE gels, transferred onto PVDF membranes, and blocked with 5% non-fat milk for 2 h. The membranes were incubated overnight at 4 °C with the following primary antibodies: Cleaved caspase-3 (CST#9661, Cell Signaling Technologies), Bcl-2 (ab196495, Abcam), Bax (CST#2772, Cell Signaling Technologies), ADFP (ab108323, Abcam), CD36 (ab133625, Abcam), and GAPDH (CST#5174, Cell Signaling Technologies). The membranes were subsequently washed and then incubated for 60 min at room temperature with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. An ECL reagent kit (Millipore, USA) and gel imaging equipment (Bio-Rad, ChemiDocTM Touch, USA) were used to detect the presence of protein bands on the membrane. The quantification of the band intensities was performed using Image Lab 5.2.1 software (BIO-RAD), and the band intensities were normalized to GAPDH.

After washed with cold phosphate‐buffered saline (PBS) for two times, the cultured cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with a cocktail of protease inhibitors. The concentrations of protein were measured using the bicinchoninic acid assay (Pierce, Appleton, WI, USA). Equivalent amounts of proteins, approximately 30 μg, were separated in 10% SDS/PAGE and electro‐transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% non‐fat milk for 2 hours at room temperature and then were incubated with the primary antibodies overnight at 4°C on a shaking table. After washed with TBST for three times, the membranes were incubated with the secondary antibody (Proteintech Group). The last analysis was conducted using ECL reagent kit (Millipore) at room temperature. GAPDH was used as internal loading control.

Total cell lysates were prepared using a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, and a cocktail of protease-inhibitors (Roche). A commercial kit for protein fractionation was purchased from Beyotime Institute of Biotechnology (cat # p0027). The proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). The proteins were detected by immunoblotting analysis using an ECL reagent kit (Millipore). GAPDH was used as an internal control. The images were captured using ChemiScope imaging system (Shanghai, China).

Proteins were extracted and immunoblotted as described previously [22 (link)]. The following primary antibodies included FAS, ATGL, glucagon-like peptide-1 receptor (GLP-1R), phospho-ERK1/2 (p-ERK1/2), total ERK1/2, P70^S6K, Bcl-2, phospho-mTOR (p-mTOR), total mTOR which were all purchased from Cell Signaling and PLIN5 from Progen Biotechnik, ACO from Abcam, CHOP from Immunoway, CPT-1 and BiP from Santa Cruz. All the secondary antibodies and internal reference GAPDH were all purchased from Santa Cruz. Immunoreactive bands were visualized with an ECL reagent kit (Millipore). Optical densities of each band were calculated and analyzed by using Image J analysis software.

Western blotting was performed as previously reported [31 (link)]. Briefly, tissues or cells were collected and lysed using RIPA buffer (P0013B, Beyotime, Biotechnology, Shanghai, China) with protease inhibitor cocktail (HY-K0010, MCE, Shanghai, China). Afterwards, samples were exposed to 5 cycles of 5 s ultrasound treatments and centrifuged at 12,000× g in a refrigerated centrifuge for 10 min. Samples were equalised according to the protein concentration and separated with SDS-PAGE. The proteins were electrically transferred to PVDF membranes (ISEQ00010, Millipore, Shanghai, China) followed by blocking the PVDF membranes for 1 h at room temperature with 5% skim (232100, BD, Sparks, MD, USA) in TBST. The membranes were incubated overnight at 4 °C with primary antibodies (A1021, anti-SERPINA3, ABclonal, Wuhan China; AC026, anti-β-ACTIN, ABclonal, Wuhan, China). The following day, membranes were washed 3 times in TBST, and incubated with HRP-conjugated secondary IgG antibody (AS014, ABclonal, Wuhan, China) for 1 h at room temperature. Before imaging, the membranes were washed with TBST 3 times and ECL reagent kit (WBKLS0500, Millipore, Shanghai, China) was used for detection of expressed proteins.