Fig. 2

PGRN processing is lysosome-dependent. a Primary Sort ^−/−, Grn ^−/−, and Psap ^−/− MEF cells, and Psap ^−/− MEF cells infected with PSAP lentivirus were labeled with [³⁵S]-methionine and [³⁵S]-cysteine for 24 h. Equal amounts of cell lysate were immunoprecipitated with a homemade rabbit anti-PGRN antibody and separated by 16% Tricine-SDS PAGE. The PGRN and PGRN-derived peptide (GRNs) signals were visualized by autoradiography. * indicates non-specific bands. b PGRN delivered from the extracellular space is processed in primary cortical neurons (DIV12). Primary cortical neurons were treated with either human PGRN (hPGRN, 1 μg/ml) alone or together with recombinant human PSAP (hPSAP, 1 μg/ml) as indicated for 16 h. The cells were harvested and proteins were separated on a 4–12% Bis-Tris gel, then blotted with goat anti-human PGRN antibodies. c Intracellular processing of PGRN is dependent on lysosomal activity. Primary MEF cells were labeled with [³⁵S]-methionine and [³⁵S]-cysteine and treated with different lysosomal inhibitors: 50 nM bafilomycin or 15 mM ammonium chloride + 100 μM chloroquine for 16 h. The cell lysates were immunoprecipitated with rabbit anti-PGRN antibodies and separated by 16% Tricine-SDS PAGE. PGRN and PGRN-derived peptides were visualized by autoradiography. * indicates non-specific bands. d Primary MEF cells were treated with different lysosomal inhibitors, as above. The cell lysates were separated on a Bis-Tris gel, then blotted with sheep anti-mouse PGRN antibodies