Fig. 4
Elevated lysosomal activity results in enhanced fast protein degradation in Grn−/−MEF. a Turnover of 35S-methionine radiolabeled proteins. MEF at 70–80% confluency were metabolically pulse-labeled with 35S-methionine/cysteine for 1 h, followed by indicated chase periods. Radioactivity of 35S- labeled proteins at chase time point 0 h was set to 100% and remaining radioactive-labeled proteins at later chase points were normalized to the initial radioactivity at time point 0 h. For statistical analysis the unpaired, two-tailed student’s t-test was used to compare Grn−/− to Grn+/+ MEF (n = 5), (*, p < 0.05). b APP holoprotein (APPholo) and C-terminal fragments (APPCTF) detected by immunoblotting of MEFpool Grn+/+ (wt) and Grn−/− (ko) lysates. The molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the blots. Bar graphs show the quantification of the blots for APPholo and APPCTF normalized to Grn +/+ (wt) as mean ± SD. c Quantification of App mRNA of MEFpool Grn−/− (ko) normalized to Grn+/+ (wt) as mean ± SD. d Ubiquitin (Ub), p62, LC3-I and LC3-II detected by immunoblotting of MEFpool Grn+/+ (wt) and Grn−/− (ko) lysates. Bar graphs show the quantification of the blots normalized to wt as mean ± SD. b-d For statistical analysis the unpaired, two-tailed student’s t-test was used to compare ko to wt cells (n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant)