CFs were lysed in 100 μL RIPA buffer (Millipore, Hertfordshire, UK) with 10 μL protease inhibitor (Sigma-Aldrich) and lysates loaded at 15 μg/lane. Proteins were separated using 10% Bis-Tris gels (Invitrogen) under reduced conditions and were transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween 20 (PBS-T) and incubated with rabbit anti-TLR4 (TP376, 1:1000, 4°C, 24 hours; AMS Biotechnology Ltd., Abingdon, UK), rabbit anti-MMP-2 (ab52756, 1:4000, 4°C, 24 hours; Abcam, Cambridge, UK), rabbit anti-MMP-9 (ab76003, 1:4000, 4°C, 24 hours; Abcam), or rabbit anti-β-actin (ab8227, 1:5000, 4°C, 24 hours; Abcam) and were washed five times in PBS-T before they were incubated with goat anti-rabbit-HRP (1:4000, room temperature [RT], 2 hours; Dako, Glostrup, Denmark). Blots were visualized using enhanced chemiluminescence (ECL Advance; GE Healthcare, Piscataway, NJ) and a molecular imager (Versadoc MP-4000; Bio-Rad). Protein expression was analyzed using image acquisition and analysis software (Quantity One; Bio-Rad).