Fig. 3
PGRN loss alters maturation and elevates activity of cathepsins in MEF. a CatD expression and maturation in MEFpool Grn+/+ (wt) and Grn−/− (ko) shown in representative immunoblots. The pro-form CatDp, single chain form CatDsc, and heavy chain form CatDhc are indicated. Bar graphs show the quantification of blots for total CatD or maturation variants normalized to Grn+/+. b CatD activity measured as cleavage of a quenched fluorogenic substrate. The increase of fluorescence signal was continuously measured and during a linear turnover time period normalized to Grn+/+. Note that extracts of CatD deficient MEF (Ctsdko) show no CatD activity and therefore confirm the specificity of the assay. c CatB expression and maturation in MEFpool Grn+/+ (wt) and Grn−/− (ko) shown in representative immunoblots. The pro-form CatBp, single chain form CatBsc are indicated. Bar graphs show the quantification of blots for total CatB or maturation variants normalized to Grn+/+. d CatB activity normalized to Grn+/+. e CatL expression and maturation in MEFpool Grn+/+ (wt) and Grn−/− (ko) shown in representative immunoblots. The pro-form CatLp, single chain form CatLsc, heavy chain form CatLhc are indicated. Bar graphs show the quantification of blots for total CatL or maturation variants normalized to Grn+/+. f CatL activity normalized to Grn+/+. g PGRN deficient MEF were stably transfected with mouse PGRN (mGrn) and low PGRN expressing single cell clones (#5, #8) were analyzed for CatD, CatB and CatL in vitro activity. Notice that very low expression of PGRN (#8) lowers cathepsin activities and thereby partially rescues the phenotype of the Grn−/− MEF, while the higher expressing clone (#5) allows a full rescue for CatB and CatL. The molecular weight standards in kilo Daltons (kDa) are indicated on the left side of the blots. All bar graphs are shown as mean ± SD. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 with ns as not significant using a-f unpaired, two-tailed student’s t-test (n = 3), g one-way ANOVA with Dunnett’s post hoc test (n = 3–6)