Immunoblot assessment of Lamp1 levels

Tissue lysates (2mg/mL) from GBA1 D409V KI hom and WT mouse brain (right hemisphere) and liver (one lobe) from the Group 1 (biochemistry) experimental cohort (N = 7 per genotype per age) described above were processed for Western blot analysis at Pfizer to determine levels of lysosomal-associated membrane protein 1 (Lamp1). Cortex was excised from the brain hemisphere and frozen. Tissue lysates were made in GBA lysis buffer composed of 10 mM Tris, pH 7.5, 250 mM sucrose, 1 mM EDTA, 0.25% Triton X-100. Western blot analysis utilized 4–12% Bis-Tris Midi gels (Thermo Fisher Scientific) with 30 μg protein loaded per well, electrophoresed at 110 V for 100 min. The resolved gel was transferred to nitrocellulose membrane (IB23001; Thermo Fisher Scientific) via dry transfer using iBlot2® Dry Blotting System (Thermo Fisher Scientific). The membranes were blocked for 1 hour with Rockland Blocking Buffer (Rockland) and then probed overnight at 4 ^oC with the following primary antibodies in Rockland Blocking Buffer with 0.1% TWEEN-20: rabbit monoclonal antibody against Lamp1 (54H11; Cell Signaling) used at 1:2,000 and mouse anti-β-actin monoclonal antibody (A2228; Sigma) used at 1:20,000. The membranes were washed 3 x 5 minutes with Tris buffered saline containing Triton-X100 (TBS-T) buffer and then incubated with 1:10,000 IRDye goat anti-rabbit 800 CW (925–32211; LiCor) and 1:10,000 IRDye goat anti-mouse 680LT (926–68020; LiCor) for 1 hour at room temperature. After incubation, the membranes were washed 3 x 5 minutes with TBS-T buffer.

The blots were visualized on LiCor Odyssey and analyzed using LiCor Odyssey software (version 4.0). Relative fluorescence units for bands associated with Lamp1 and β-actin were calculated by the LiCor Odyssey software for statistical analysis. Lamp1 levels were normalized to β-actin levels from the same tissue sample. Two-Way ANOVA tests were performed to understand the effect of genotype and age. Bonferroni post hoc tests were used to understand the significance of the individual comparisons. Individual data points with mean and standard error of the mean are represented on each graph.