Epr4716 2

For liver histology, after mouse euthanasia, one lobe of the liver was snap-frozen in isopentane (−160°C) previously chilled in liquid nitrogen. Serial 12-μm cross-sections were cut in a Leica CM3050 S cryostat (Leica Biosystems). Two to four mice representative of the tested cohort were randomly selected. Liver sections were fixed with 4% paraformaldehyde (PFA), blocked and permeabilized with 10% normal goat serum (NGS) in 0.1% Triton X-100 for 3 hr at room temperature and stained with the primary anti-hGAA antibody (rabbit monoclonal, clone EPR4716(2), Abcam) overnight at 4°C. After washing, the sections were incubated with anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific) combined with DAPI. Representative images were acquired with an SP8 confocal microscope (Leica).

HuH7, C2, and NSC34 cell lysates were prepared using 10 mM PBS (pH 7.4) containing 1% of Triton X-100 and protease inhibitors (Roche Diagnostics). Western blot on mouse plasma was performed on samples diluted 1:4 in distilled water. Homogenates of mouse tissues were prepared as indicated for GAA activity. Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific). SDS-PAGE electrophoresis was performed in a 4%–12% polyacrylamide gel. After transfer, the membrane was blocked with Odyssey buffer (LI-COR Biosciences) and incubated with an anti-GAA antibody (rabbit monoclonal, clone EPR4716(2), Abcam), anti-EGFP (mouse monoclonal, sc-9996, Santa Cruz Biotechnology), anti-tubulin (mouse monoclonal, clone DM1A, Sigma-Aldrich), or anti-Gapdh (rabbit polyclonal, PA1-988, Thermo Fisher Scientific). The membrane was washed and incubated with the appropriate secondary antibody (LI-COR Biosciences) and visualized with the Odyssey imaging system (LI-COR Biosciences). For western blot quantification, we used either ImageJ or Image Studio Lite 4.0. The quantification of the hGAA protein bands in mouse tissues was normalized using either tubulin or Gapdh bands. The quantification of the hGAA protein band in plasma was normalized using a non-specific band detected by the anti-hGAA antibody in mouse plasma (used as a loading control).