The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Teoh, Kim-Tat; Siu, Yu-Lam; Chan, Wing-Lim; Schlüter, Marc A.; Liu, Chia-Jen; Peiris, J. S. Malik; Bruzzone, Roberto; Margolis, Benjamin; Nal, Béatrice

Approximately 2.5 × 104 MDCKII cells were seeded onto 12-mm transwell membrane filters (Corning, Lowell, MA, USA) and cultured in culture medium containing 1.8 mM calcium until confluency. The monolayers were washed three times with 1X PBS and maintained overnight in low calcium medium [S-MEM, 10% dialyzed FBS (Invitrogen), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.292 mg/ml L-glutamine] containing 5 μM calcium to dissociate intercellular junctions. On the following day, the low calcium medium was replaced with prewarmed normal medium (1.8 mM calcium), and transepithelial electrical resistance (TER) was measured at the specified time points post-calcium switch (t = 0, 1, 2, 4, 5, 6, 8, 17, 24, 120 h). In brief, TER was determined as described previously (Straight et al., 2004) using a Millicell-ERS volt-ohm meter (Millipore, Billerica, MA, USA).