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

The pulled down and coimmunoprecipitated proteins were solubilized in LDS sample buffer (Invitrogen), 10 mM DTT, boiled at 95°C for five minutes, and separated by electrophoresis using NuPAGE Novex 4–12% Bis-Tris Mini gel and subsequently transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked overnight at 4°C in 10% skimmed milk prepared in 1X phosphate buffered saline (PBS), 0.1% Tween-20. To detect Flag-PALS1 membranes were hybridized with mouse IgG1 monoclonal anti-Flag M2 HRP-conjugated antibody; E, HA-E (wt), and HA-E (ΔPBM) proteins were detected by hybridization of the membranes with primary antibodies against rabbit anti-E and mouse IgG1 monoclonal anti-HA tag (Sigma Aldrich), respectively, followed by HRP-conjugated secondary antibodies. Antibody solutions were prepared in 5% skimmed milk, 1X PBS, 0.1% Tween-20. To visualize the protein bands, membranes were hybridized with ECL Western blotting detection reagent (Amersham Biosciences).