Ab76493

The total protein was extracted from tissues and cells, and the protein concentration was measured using a BCA kit (Thermo Fisher Scientific, USA). A total of 30 μg of total protein was subjected to polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Amersham, USA). The membrane was blocked with 5% skim milk powder at room temperature for 1 h and incubated at 4 °C overnight with rabbit antibodies against TLR4 (2 ug/mL, ab13556, Abcam, UK), IRF3 (1 ug/mL, ab68481, Abcam, UK), phosphorylation of IRF3 (1 mg/mL, ab76493, Abcam, UK), YAP (10 mg/mL, ab76252, Abcam, UK), CDX2 (10 mg/mL, ab76541, Abcam, UK), E-cadherin (10 mg/mL, ab40772, Abcam, UK), N-cadherin (1 mg/mL, ab18203, Abcam, UK), Bax (1: 1000, ab32503, Abcam, UK) and Bcl-2 (1: 1000, ab32124, Abcam, UK). The membrane was washed 3 times with PBST (PBS buffer containing 0.1% Tween-20), 10 min each times. Subsequently, horseradish peroxidase-labeled secondary goat anti-rabbit IgG (10 mg/mL, ab6721, Abcam, UK) was added to the membrane for incubation at room temperature for 1 h. The membrane was washed 3 times with PBST buffer for 10 min each. After scanning and development with an optical luminometer (GE Healthcare, USA), the protein band intensities were performed using Image Pro Plus 6.0 software (Media Cybernetics, USA), followed by analysis of the relative protein expression.