Quantity one system image analyzer

Cell lysates were scraped and dissolved in RIPA buffer (Beyotime, China) containing PMSF (1:100, Beyotime, China) on ice, followed by centrifugation at 12,000 g for 15 min to collect the protein in the supernatant. The protein concentration was standardized and mixed with Laemmli Sample Buffer (#4006028, Bio-Rad, United States) and DTT, followed by separation on a 10% discontinuous SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Merck Millipore, GER). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h, and then incubated with specific primary antibodies overnight at 4°C. After that, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG for 1 h at room temperature. Band densitometry was performed using the Quantity One System image analyzer (Bio-Rad, United States). The antibodies used in this study included ATG5 (1:1,000, ab108327, Abcam, United States), BECLIN1 (1:1,000, ab207612, Abcam, United States), LC3 (1:1,000, 14600-1-AP, Proteintech, China), P62 (1:1,000, ab109012, Abcam, United States), and β-Actin (1:1,000, Cell Signaling Technology, United States). All original gel images generated from Western blotting are included in Supplementary Figure S9.

ADO2-iPSCs and NC-iPSCs were homogenized in RIPA buffer (cat. no. KGP702–100; Changchun Keygen Biological Products Co., Ltd.) containing protease inhibitor at 4 °C, and then centrifuged at 16,000 x g for 15 min at 4 °C to extract soluble protein. Protein extracts were quantified using the BCA method and loaded on 12% SDS-gels (20 μg per lane), resolved using SDS-PAGE, and then transferred to nitrocellulose membranes. Following blocking (5% skim milk) for 2 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against CLC-7 (1:1000), or α-tubulin (1:2000). Subsequently, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:1000) at room temperature for 2 h. Signals were visualized using a Quantity One system image analyzer (Bio-Rad Laboratories, Inc.) and densitometry analysis was performed using Image J.

The protein was extracted from liver tissues and cell harvests. Equal amounts of extracted protein (approximately 20 μg) were separated by using SDS‐PAGE gel and transferred onto polyvinylidene fluoride membrane (Bio‐Rad, CA). After 2 hours of blocking with 5% BSA, the membranes were incubated with primary antibodies (GAPDH, LC3, p62, cIAP1, cIAP2, XIAP, and survivin) at 4°C overnight, followed by incubation with secondary antibodies at 20‐25°C for 2 hours. Antibodies against GAPDH, cIAP1, cIAP2, LC3, and p62 were obtained from CST, while antibodies against XIAP and survivin were obtained from Abclonal. The immunoblots were visualized using electrochemiluminescent reagents (GE Healthcare, IL) following the manufacturer's instructions, and quantified using Quantity One System image analyzer (Bio‐Rad).