Odyssey laser imaging system

Proteins were separated by 10% SDS-PAGE and transferred to NT nitrocellulose membranes (66485, BIOTRACE). After blocking with 5% milk, membranes were incubated with anti-FLAG (1:5,000; F1804; Sigma-Aldrich) and anti-OFD1 (1:30,000) for 1 hour at room temperature. After washing with PBS 3 times, the membranes were incubated with secondary antibodies for 1 hour at room temperature. The ChemiDoc Touch Imaging System (Bio-RAD) and Odyssey Laser Imaging System (LI-COR) were used for image acquisition.

The GCs were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1% (v/v) PMSF (Boster, Wuhan, China) for 30 min on ice. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. Protein concentrations were quantified using a BCA protein assay kit (Boster, Wuhan, China). The proteins (10 µg) were separated using 8% or 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Boster, Wuhan, China). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature. Then, the membranes were incubated with rabbit polyclonal primary antibodies against NICD2 (1:1000, Cell Signaling Technology, Boston, MA, USA), β-catenin (1:1000, Cell Signaling Technology, Boston, MA, USA), Bax (1:500, Bioss, Beijing, China), and caspase-3 (1:500, Bioss, Beijing, China) overnight at 4 °C. After washing with TBS, the membranes were incubated with IRDye^® 800CW goat anti-rabbit IgG secondary antibody (1:18,000, LI-COR Biosciences, Shanghai, China) for 1 h at room temperature. The blots were imaged using an Odyssey laser imaging system (LI-COR Biosciences, Lincoln, NE, USA) and quantified using ImageJ software version 1.8.0. The protein levels were normalized to β-actin (1:10,000, BioWorld, Nanjing, China).