A8529

We utilized the following antibodies to perform Western blot analysis. The primary antibodies were anti-4-HNE (ARG23717, Arigo Biolaboratories, HSZ, Taiwan), anti-xCT (26864–1-AP, Proteintech, Rosemont, IL, USA), anti-GPX4 (ARG41400, Arigo Biolaboratories, HSZ, Taiwan), anti-GAPDH (ARG10112, Arigo Biolaboratories), anti-β-actin (NB600-501, Novus Biologicals, TPE, Taiwan), anti-catalase (219010, Merck, DA, Germany), anti-SOD2 (# 06–984, Merck), anti-SOD1 (ab51254, Abcam, EN, UK), anti-ULK1 (A8529, ABclonal, Woburn, MA, USA), anti-ND-1 (A5250, ABclonal), anti-UQCRC2 (A4181, ABclonal), anti-p62 (66184–1-Ig, Proteintech), and anti-Lamp2 (ab125068, Abcam). The secondary antibodies were anti-mouse IgG (7076, Cell Signaling, Danvers, MA, USA) and anti-rabbit IgG (7074, Cell Signaling).

The protein was lysed using RIPA lysis buffer, and BCA method was adopted to determine the protein concentration. The precast gel was removed from the refrigerator at 4°C and put into the electrophoresis tank. Subsequently, 500 μg of total protein was added to each sample and mixed by the additional of 5× SDS loading buffer at 4:1 ratio. After mixing, the protein concentration was set at 3.3 μg/μL. The metal bath was heated at 100°C for 6 min to denature the protein. The denatured total protein was taken 60 μg for loading. After running at 80 V through the concentrated glue, the voltage was adjusted to 120 V, and waited until the bromophenol blue reached the bottom of the rubber sheet without spilt out. After transferring the membrane, 5% skimmed milk blocking solution was supplied for blocking at room temperature for 1 h. Primary antibody was added for incubation overnight at 4°C, washed 3 times with TBST, 10 min each time, added secondary antibody and incubated at room temperature for 1 h, washed with TBST 3 times, 10 min each time, and exposed using ECL exposure liquid. The antibodies used were: FEZ1 (A15362, abclonal, China), 1:500; SCOC (PH309356, ORIGEN, United States), 1:500; ULK1 (A8529, abclonal, China), 1:500; NBR1 (A3949, abclonal, China), 1:500; GAPDH (A19056, abclonal, China), 1:2000; and secondary antibody (AS014, abclonal, China), 1:1000.

The collected tissues and cells were lysed with RIPA lysis buffer (Beyotime) to extract the proteins. Next, 50 μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked in 5% non-fat dried skimmed milk (Beyotime) for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Finally, the membranes were incubated in blocking buffer with secondary antibody (1:2500; AntGene, Wuhan, China) for 2 h before detection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. Primary antibodies against the following targets were employed: UCP1 (ab10983, 1:1000), GAPDH (60004-1-Ig, 1:1000; Proteintech), NGAL (ab63929, 1:1000; Abcam), Bax (50599-2-Ig, 1:1000; Proteintech), Bcl-2 (12789-1-AP, 1:1000; Proteintech), cleaved caspase-3 (9664, 1:1000; Cell Signal Technology, Danvers, MA, USA), P-AMPK (AP0116, 1:1000; ABclonal), AMPK (A17290, 1:1000; ABclonal), P-ULK1 (AP0736, 1:1000; ABclonal), ULK1 (A8529, 1:1000; ABclonal), and P62 (A11250, 1:1000; ABclonal). The densitometric analysis of western blot images was performed by ImageJ2x. Statistical analyses were performed using SPSS Statistics 22.0 (IBM SPSS, Chicago, IL) and Excel 2016 (Microsoft).