Am4300

Cell lysates were prepared in RIPA buffer, subjected to SDS-PAGE (Thermo Fisher Scientific), and transferred to PVDF membranes (Bio-Rad) at 100 V for 1 h. After blocking, the membranes were incubated with 5% nonfat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) containing antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), USF1 (Proteintech, 22327–1-AP, 1:2000), USF2 (Novus, NBP1-92649, 1:2000), and CTCF (Abcam, ab70303, 1:1000) at 4 °C for 12 h with gentle shaking. The membranes were washed three times for 30 min and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 h at room temperature. The blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences).

Cells lysate was prepared by using RIPA buffer followed with SDS-PAGE (Thermo Fisher Scientific) and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at constant 100 V for 1 hr. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 hr at room temperature, the membrane was incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), MYC (Cell Signaling Technology, #9402, 1:1,000), USF2 (Novus, NBP1-92649, 1:2,000), USF1 (Proteintech, 22327–1-AP, 1:2,000), GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), Vinculin (Proteintech, 26520–1-AP, 1:2,000) and CTCF (abcam, ab70303, 1:1,000) at 4°C for 12 hr with gentle shaking. Membranes were washed three times for 30 min and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 2 hr at room temperature. Blots were washed with TBS-T three times for 30 min and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocol.