Fig. 3

Asparagine endopeptidase (AEP) is a PGRN protease that liberates granulin F. a-b, Differentiated SH-SY5Y cells were treated with siRNA against AEP or scramble control for 72 h. Cells were lysed and western blotting performed for endogenous AEP, PGRN or Gran-F as indicated. Mature AEP, PGRN and Gran-F were significantly decreased in AEP siRNA treated cells (*p < 0.04, **p = 0.0010) relative to actin. Unpaired student’s t-test, error bars represent mean with standard deviation, n = 3. c, HEK293FT cells were transiently transfected with FLAG-tagged AEP (AEP FLAG or AEP OE) or FLAG alone (mock) for 24 h and cells lysates were probed for endogenous PGRN and Gran-F. AEP expression was confirmed using an anti-FLAG antibody. Each biological replicate was run on a separate western blot and normalized to actin. d-e, Quantification of c showing that overexpression of AEP decreased endogenous PGRN and increased Gran-F levels. Paired t-test analysis, p values are indicated, error bars represent mean with standard deviation, n = 4. f, HEK293FT cells were transiently transfected with FLAG-tagged CTSL (CTSL FLAG) or FLAG alone (mock) for 24 h and cells lysates were probed for endogenous PGRN and Gran-F. CTSL expression was confirmed using an anti-FLAG antibody. Each biological replicate was run on a separate western blot and normalized to actin. g-h, Quantification of f showing that overexpression of CTSL decreased endogenous PGRN but did not alter Gran-F levels. Paired t-test analysis, p values are indicated, error bars represent mean with standard deviation, n = 4