Fig. 5: R702W SorCS2 is susceptible to aberrant proteolytic cleavage.

A western blot of elution profile from immunoaffinity chromatography purification of WT and R702W SorCS2 ECD (n = 1). B sucrose gradient centrifugation of HEK293T cells transfected with WT or R702W SorCS2 (n = 3). Multiple fragments of R702W SorCS2 in high-density fractions are indicated. C pulse-chase experiment showing increased release of R702W SorCS2 ECD. Transfected HEK293T cells were biolabeled and SorCS2 immunoprecipitated from culture supernatant at indicated time points and subjected to SDS-PAGE and autoradiography. Western blot (n = 3) of stead-state level of SorCS2 in cells is also shown. D quantification of C. Values are normalized to WT SorCS2 at 20 h and shown as mean ± SEM. Data analyzed by unpaired two tailed Student’s t test. E representative western blot of released SorCS2 ECD immunoprecipitated from culture supernatant of transfected HEK293T cells in the presence or absence of 30 µM galardin (gal). F quantification of E. Shown is mean ± SEM (n = 3). Data analyzed by unpaired two tailed Student’s t test. G and H a SorCS2 specific ELISA was established (G) in order to measure serum levels of SorCS2 ECD in a control group and a member of the affected family (patient X) (n = 1). Median with interquartile range is shown for the control group.