Adam10

Cells were lysed with RIPA lysis buffer (Sangon Biotech Co., Ltd.) supplemented with protease and phosphatase inhibitors. Total protein was quantified using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein (30 µg/lane) were separated via 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with TBS-T (0.05% Tween-20) containing 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated at 4°C overnight with primary antibodies targeted against: β-actin (1:1,000; cat. no. 3700T; Cell Signaling Technology), CXCL16 (1:1,000; cat. no. 60123-1-Ig; Proteintech), ADAM10 (1:1,000; cat. no. BS60338; Bioworld Technology.) and ACTN4 (1:1,000; cat. no. BS90025; Bioworld Technology). Following primary incubation, the membranes were incubated with horseradish peroxidase-conjugated Affinipure Goat Anti-Rabbit IgG (H+L; 1:5,000; cat. no. SA00001-2; Proteintech) or Affinipure Goat Anti-Mouse IgG (H+L; 1:5,000; cat. no. SA00001-1; Proteintech) secondary antibodies for 1 h at room temperature. Protein bands were visualized using Western Bright ECL (Thermo Fisher Scientific, Inc.). Protein expression was quantified using Image Lab software (version 4.1; Bio-Rad Laboratories) with β-actin as the loading control.