β actin

RT–PCR and western blot analysis were performed as described previously [28 (link)]. The following primary antibodies were used for the immunoblotting assay: β-actin (AbFrontier, Seoul, Korea), ERRγ (Perseus Proteomics, Tokyo, Japan), and fibrinogen (Dako, Carpinteria,CA, USA). All primer sequences are described in S1 Table.

Crotonaldehyde, Compound C, and bafilomycin A1 were obtained from Sigma (St. Louis, MO, USA). SB203580 was purchased from Calbiochem (La Jolla, CA, USA). The following antibodies were used for the present study: p38, phospho-p38, AMPK, phospho-AMPK (Cell Signaling Technology, Beverly, MA, USA), SQSTM1/p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), beclin1 (BD Biosciences, San Jose, CA, USA), and β-actin (AbFrontier, Seoul, Korea). All other chemicals and reagents used were of analytical grade.

The tissues were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Translab, Sacramento, CA, USA) for 20 min on ice. We used a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Waltham, MA, USA) to assess the protein concentration of the protein extract. Protein (70 μg) was separated on 12% SDS-PAGE, and then transferred onto PVDF (Millipore) activated by absolute methanol. The PVDF membrane was blocked with 5% skim milk (BD Bioscience, San Diego, CA, USA) in 1X TBS-T buffer for one hour at room temperature. Membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies used are as follows: Pclo (Abcam, ab20664; diluted 1:1000), Clstn3 (Proteintech, 13302-1-AP; diluted 1:1000), Bdnf (Abcam, ab108319; diluted 1:1000), and β-actin (AbFrontier diluted 1:5000). After primary antibody incubation, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000 dilution) for two hours at room temperature. The protein bands were detected using an ECL solution (Thermo Fisher Scientific) and iBright CL1000 imaging system (Invitrogen) according to the manufacturer’s instructions. Protein levels were normalized to β-actin protein levels.

For immunoblotting, samples were lysed on ice using lysis buffer and boiled for 5 min in Laemmli sample buffer. Samples were loaded into SDS-polyacrylamide gels under denatured conditions and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with skim milk at RT for 1 h and incubated with the following primary antibodies at 4 °C overnight (O/N): β-actin (Abfrontier, Seoul, Republic of Korea, LF-PA0207, 1:2000), t-EGFR (Abcam, Waltham, MA, USA, ab52894, 1:10,000), p-EGFR (Abcam, ab40815, 1:5000), t-HER2 (Santa Cruz Biotechnology, CA, USA, sc33684, 1:5000), p-HER2 (CST, Danvers, MA, USA, 2241S, 1:1000), and CD11b (Abcam, ab52478, 1:1000). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (CST) at RT for 1 h. Protein signals were developed using ECL^TM prime reagent (GW Healthcare, Bucks, UK).

The harvested kidney tissue was homogenized and lysed in a pro-prep extraction solution (iNtRON Biotechnologist, Seong-nam, Korea) followed by protein quantitation with the Bradford method^{34 (link)}. In addition, nucleus and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific, Rockford, IL, USA). Lysates were fractioned on 10–15% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4 °C with primary antibodies against HMGB1 (1:1000, #ab18256, Abcam, Cambridge, MA), cleaved caspase-3 (1:1000, #9664, Cell signaling, MA, USA) β-actin (1:10000, #LF-PA0207, AbFrontier, Seoul, South Korea) and LaminB1 (1:1000, #12586, Cell signaling, MA, USA). After the membranes were washed three times in 1xTBS-T for 15 min each, they were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG-HRP, 1:10000 #SA002-500, GenDepot, Houston, TX) for 1 h at room temperature. Then the membranes were washed three times in 1xTBS-T for 15 min again. The blotted membranes were visualized by ECL reagents and exposed to X-ray film. The results were normalized to the β-actin and LaminB1 loading control and band density was measured using Image J software (National Institutes of Health, Bethesda, MD, https://imagej.nih.gov/ij)^{34 (link)}.