Flag tag (flag)

QBC939 or RBE cells at 80-90% confluency in two 10-cm diameter dishes were scraped on ice into cold phosphate-buffered saline (PBS), pooled and lysed with 1 ml of Pierce™ IP lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with protease inhibitors (Roche Diagnostics). Protein G magnetic beads (Bio-Rad Laboratories, Inc.) were washed three times with PBS-0.1% Tween-20 and incubated with 3 µg of antibody in a final volume of 200 µl for 30 min at room temperature. The beads were washed three times and incubated overnight at 4°C. The beads were washed again, and 30 µl of 1X loading buffer was added. The slurry was incubated at 100°C for 10 min, and the beads were discarded. The supernatant was collected for western blotting conducted as described below. The following antibodies were used: GATA6 (cat. no. 5851; Cell Signaling Technology, Inc.); LOXL2 (cat. no. MAB2639; R&D Systems, Inc.); Flag (cat. no. 14793; Cell Signaling Technology, Inc.); rabbit IgG (cat. no. bs-0295P; BIOSS), and mouse IgG (cat. no. bs-0296P; BIOSS). HRP-conjugated VeriBlot for IP (1:1,000; cat. no. ab131366; Abcam) was used as the secondary antibody, and the membrane was incubated for 2 h at room temperature.