Western blotting

Cells were lysed on ice for 20 min with RIPA buffer and the protein concentration was assessed using a BCA kit (Beyotime Institute of Biotechnology, Haimen, China). The cell lysates (Si-1, Si-N and N) and immunocomplexes were resolved using 12% SDS-PAGE and electronically transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking at 4°C overnight with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20, the membranes were incubated with primary antibodies against ABCE1 (dilution, 1:5,000; cat. no. ab185548; Abcam), GAPDH (dilution, 1:10,000; cat. no. AP0063; Bioworld Technology, Inc., St. Louis Park, MN, USA), cyclin-dependent kinase inhibitor 1A (dilution, 1:3,000; cat. no. ab109520; Abcam), caspase 3 (dilution, 1:5,000; cat. no. ab13847; Abcam), MDM2 proto-oncogene (dilution, 1:3,000; cat. no. ab38618; Abcam) and B cell leukemia/lymphoma 2 (dilution, 1:500; cat. no. ab32124; Abcam) at 4°C overnight. Subsequently, the membranes were washed three times with TBST buffer. The membranes were then incubated with anti-rabbit (dilution, 1:10,000; cat. no. ab150077; Abcam) or anti-mouse (dilution, 1:10,000; cat. no. ab150113; Abcam) secondary antibodies conjugated to horseradish peroxidase (EMD Millipore) at room temperature for 2 h. The bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.) Densitometry was calculated using Quantity One software 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).