Anti pgrnfl

Total protein concentration was measured in LCL lysates and media by the BCA assay (Pierce-Thermo, Rockford, IL, USA). Cell lysates and media were normalized based on total protein concentration prior to immunoblotting. The samples were incubated in Laemmli buffer supplemented with β-mercaptoethanol, boiled, and run on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The goat polyclonal antibody raised against full-length PGRN (anti-PGRN^FL, 1:300 dilution; R&D Systems, Minneapolis, MN, USA) and the rabbit polyclonal antibody against human progranulin (1:500 dilution; Genetex, Irvine, CA USA) were used. An antibody against β-tubulin (dilution 1:20,000; Sigma-Aldrich, St Louis, MO) was used in parallel for further normalization.

To measure the rate at which elastase cleaves PGRN (WT, C139R, C521Y, A199V, C105Y, R298H), serum-free culture media was collected and treated with 1U elastase diluted in 100 mM Tris-HCl and 960 mM NaCl to achieve a final concentration of 5U/ul. Elastase-treated media was then incubated at 37°C for 5, 15, 30, 60, and 90 minutes. PGRN and GRN protein fragments were assessed using SDS-PAGE and immunoblotting as described above. Immunoblots were probed with the anti-PGRN^C-term antibody or, in parallel experiments performed under non-reducing conditions, with a goat polyclonal antibody raised against full-length PGRN (anti-PGRN^FL, 1:1000 dilution; R&D Systems, Minneapolis, MN, USA).