Author: 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
Title: The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis Document date: 2010_11_15
ID: ufw13pjx_24
Snippet: HEK 293T cells were transfected with pcDNA-Flag-PALS1, pcDNA-E (wt), pcDNA-HA-E (wt), or pcDNA-HA-E (ΔPBM) constructs and lysed forty-eight hours post transfection as described above. Agarose bead-conjugated anti-Flag M2 affinity gel suspension was washed four times in Tris-buffered saline buffer (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH7.4) as described by the manufacturer (Sigma-Aldrich). For precipitation of Flag-PALS1 protein, 40 μl agarose bea.....
Document: HEK 293T cells were transfected with pcDNA-Flag-PALS1, pcDNA-E (wt), pcDNA-HA-E (wt), or pcDNA-HA-E (ΔPBM) constructs and lysed forty-eight hours post transfection as described above. Agarose bead-conjugated anti-Flag M2 affinity gel suspension was washed four times in Tris-buffered saline buffer (TBS, 50 mM Tris-HCl, 150 mM NaCl, pH7.4) as described by the manufacturer (Sigma-Aldrich). For precipitation of Flag-PALS1 protein, 40 μl agarose beads were incubated overnight at 4°C with 600 μl of cell lysate, centrifuged, washed five times with TBS buffer and coimmunoprecipitated proteins were analyzed by electrophoresis and immunoblotting.
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