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

For MDCKII cysts, the medium was drained off, followed by three extensive washes in 1X PBS. Next, cysts were fixed with 4% PFA, 1X PBS, 0.02% sodium azide for one hour, permeabilized with 0.1% SDS, 1X PBS, 0.02% sodium azide for 30 min at room temperature and blocked as described above. MDCKII cysts were incubated with primary antibodies diluted in 2% NGS, 1X PBS, 0.02% sodium azide for 48 h and then extensively washed (3×) in the same medium. Alexa Fluor-conjugated secondary antibodies were added for 24 h at 4°C in a humidified chamber. For MDCKII cells grown on transwell membrane filters, cells were fixed, permeabilized, and immunostained as described above with minor modifications. Cell nuclei were labeled with 4′,6′–diamidino-2-phenylindole (DAPI). BIOCHIPs and Vero E6 cells were then mounted with anti-fading agent Mowiol 4–88 reagent (Merck KGaA. Darmstadt, Germany). Images were acquired using ZEISS LSM 510 Axiovert 200M inverted confocal microscope (Core Imaging Facility of The University of Hong Kong) and Olympus Fluoview FV500 confocal microscope system (The Morphology and Image Analysis Core -MIAC- of the University of Michigan Diabetes Research and Training Centre).