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Figure 1


Fig. 1. Phosphorylation of paracellin-1 by cAMP. (A-F) Whole membrane fractions were prepared from MDCK cells expressing FLAG (Mock) or FLAG-tagged PCLN-1 (PCLN-1). The fractions (30 µg) were applied to SDS-polyacrylamide gel and then immunoblotted with antibodies raised against PCLN-1 (A), FLAG (B), occludin (C), claudin-1 (D), claudin-4 (E) or ZO-1 (F). To quantify the expression levels of these proteins, the amounts of occludin, claudin-1, claudin-4 and ZO-1 in the mock cells were normalized as 100%. In the PCLN-1-expressing cells, the relative expression levels of occludin, claudin-1, claudin-4 and ZO-1 were 98.6±8.5% (n=4), 104±4.1% (n=6), 102±9.0% (n=6) and 98.2±2.5% (n=4), respectively. (G-J) MDCK cells expressing FLAG-tagged PCLN-1 were treated with 50 µM H-89, 10 µM PKI or 50 µM DDA for 1 hour. When used, 100 µM 8-Br-cAMP (cAMP) was treated for 1 hour after the addition of DDA. Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG antibody (G,I). The whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody. The immune pellets were immunoblotted with anti-phosphoserine (P-Ser) antibody (H,J).





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