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Figure 6


Fig. 6. Dysfunction of PCLN-1 by the S217A mutation. (A) Schematic description of the GST-fused cytoplasmic C-terminal domain of PCLN-1. (B) Proteins were fractionated on gels and stained with Coomassie Brilliant Blue (CBB) for the detection of GST proteins. The proteins bound to beads were incubated with the activated PKA subunit, and were then immunoblotted with anti-phosphoserine antibody (P-Ser). (C-E) Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG (C), anti-claudin-1 (D) or anti-claudin-4 antibodies (E). As compared with cells expressing WT PCLN-1, the relative expression levels in cells expressing the S217A mutant PCLN-1 were 102±6.0% (claudin-1) and 97.1±9.0% (claudin-4), respectively. (F,H) Whole membrane fractions (500 µg) were incubated with protein G-Sepharose and anti-FLAG antibody to immunoprecipitate (IP). The immune pellets were immunoblotted (IB) with (F, upper) anti-phosphoserine (P-Ser), (F, lower) anti-PCLN-1, or (H, upper) anti-ZO-1 antibodies. Whole membrane fractions (30 µg) were immunoblotted with anti-FLAG or anti-ZO-1 antibodies (H, lower). (G) The Triton X-100-soluble (60 µg) and -insoluble fractions (30 µg) were immunoblotted with anti-FLAG-antibody. (I-K) TER, FITC-dextran-4k flux and Mg2+ transport were measured in MDCK cells expressing FLAG (Mock), FLAG-tagged WT PCLN-1 (WT), or FLAG-tagged S217A mutant PCLN-1 (S217A). **P<0.01. NS, not significant.





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