Western Blot Analysis of KCTD1 Signaling

The detailed protocol is provided in [20 (link)]. The antibodies used were Anti-KCTD1 (LS-C260993, LS-Bio, Seattle, WA, USA), Anti-Flag (MA1-91878, Thermo Fisher), anti-IKβα (662402, Biolegend, USA), anti-β-catenin (Sc-7963, SantaCruz), and anti-β-actin (ab11004, Abcam, UK) as loading controls. Briefly, 30 μg of protein extracts from each sample were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBS-T (Tris-buffered pH8/0.15% Tween 20) at room temperature for 1 h. Then, they were incubated with primary antibodies diluted in 5% non-fat milk in TBS-T (according to manufacturer instructions) overnight at 4 °C. After three washes in TBS-T, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. The signal intensity was visualized using Clarity Max Western ECL Substrate (BioRad cat#1705062) and acquired using the ChemiDoc Imaging system (Bio-Rad, USA). Semi-quantitative densitometric analysis was performed using Image Lab 5.2.1 (Bio-Rad). Protein band normalizations were conducted dividing the signal of the protein of interest to the signal of β-Actin.