HCECs were pretreated with U0126 (Cell Signaling Technology, Beverly, MA), EGTA, nifedipine, or phosphate-buffered saline (PBS) and then treated with anti-asialoGM1 antibody. After treatment, cells were washed twice with ice-cold PBS (pH 7.4) and lysed for at least 5 minutes on ice in whole cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, and 1 mM Na3VO4), with a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamide, 10 μg/mL leupeptin, and 10 μg/mL aprotinin). Cells were harvested by scraping followed by sonication (three bursts at 40 mV for 10 seconds), and cell lysates were clarified by centrifugation at 4°C for 15 minutes at 13,000 rpm. Protein concentrations of the lysates were measured with a bicinchoninic acid assay (Micro BCA protein assay kit; Pierce Biotechnology, Rockford, IL), and equal amounts of total protein were fractionated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. After semidry transfer to polyvinylidene difluoride (PVDF) membranes, the membranes were blocked with PBS containing 0.1% Tween-20 (PBS-T) and 5% nonfat dry milk (Carnation, Nestlé USA, Glendale, CA) for 1 hour at room temperature and then incubated with specific antibodies for 2 hours at room temperature or overnight at 4°C. The antibodies used were: anti-asialoGM1 (1:50,000–1:100,000 dilution; Wako Chemical, Richmond, VA), anti-TLR5 (1:1000), anti-phospho-ERK1/2 (1:1000), and anti-ERK1/2 (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA). After primary antibody incubation, PVDF membranes were washed three times with 5% nonfat dry milk in PBS-T and then incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000–1:10,000 dilution; Santa Cruz Biotechnology) for 1 hour at room temperature. After successive washings, bound antibodies were visualized with an enhanced chemiluminescence system (ECL Plus Western Blot detection system; GE Healthcare, Piscataway, NJ).