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Fig. 8. Degradation of dephosphorylated PCLN-1 in the lysosome. (A) MDCK cells expressing WT PLCN-1 were treated with 50 µM H-89, 50 µM H-89 plus 10 µM lactacystin, or 50 µM H-89 plus 100 µM chloroquine for 3 hours in the presence of 10 µM cycloheximide. (B) MDCK cells expressing the S217A mutant PLCN-1 were treated with 10 µM lactacystin or 100 µM chloroquine for 3 hours in the presence of 10 µM cycloheximide. Whole membrane fractions (30 µg) were immunoblotted with anti-ZO-1 (A,B, upper) or anti-FLAG antibody antibodies (A,B, middle). The 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 anti-ubiquitin antibody (Ub; A,B, lower).
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