Association between CLN5 and PPT1/CLN1. (A) COS-1 cells were transiently transfected with PPT1/CLN1 together with mCLN5-myc/his or ORP1L-his. Co-immunoprecipitation was performed with a his-specific antibody, followed by Western blot detection for PPT1/CLN1. CLN5 was found to bind specifically with PPT1/CLN1, confirming the interaction between the proteins. (B) RNA was extracted from the whole cortical area of four Cln5-/- mice, four Cln1-/- mice and four of their respective wt littermates and pooled together into two wild-type and two knock-out samples. Real time RT-PCR was then performed to measure expression of the two NCL proteins. The value for the wt gene expression was set to 1 and Cln5 and Ppt1 expression values are shown relative to that. Expression of CLN1 (black columns) was over two-fold up-regulated in the 4-month-old Cln5-/- mice compared to that of the wt. The expression of CLN5 (gray columns) was only slightly altered in Cln1-/- mice of the same age.