Ab55080

Cells were lysed in cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) sodium deoxycholate, 1% (v/v) NP-40, pH 8) with protease and phosphatase inhibitor for 30 min. The lysates were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatant protein was quantified using BCA assay. Total protein was normalized, mixed with 1x SDS-PAGE loading buffer, denatured at 95°C for 5 min and resolved on an SDS-polyacrylamide gel, and transferred to PVDF membrane. For dot blots, conditioned media was blotted on a nitrocellulose membrane without boiling. Membranes were blocked with 5% bovine serum albumin (Sigma). Blots were probed with primary antibodies to LAMP1 (abcam ab108597), GCase (abcam ab55080), pT73-Rab10 (abcam ab230261), Rab10 (cell signaling 8127S), pT72-Rab8a (abcam ab230260), Rab8a (abcam ab188574), pS935-LRRK2 (abcam 133450), LRRK2 (clone, 8629). αSyn oligomer (abcam ab209538). Secondary antibodies conjugated to horseradish peroxidase were used for detection using autoradiography.

Fibroblasts were trypsinized and resuspended in 1 ml PBS. Each sample was then treated with 100 µl lysis buffer with protease inhibitors (1% Triton™ X-100 with 1 µg/ml leupeptin, 1 µM PMSF, 1 µg/ml pepstatin, 1 μM NaV). Cell lysates were then centrifuged at full speed for 10 min and the supernatant transferred to fresh tubes. Lysates containing 30 µg of protein were then electrophoresed on a 4–12% NuPage gel (Invitrogen). Proteins were transferred to a PVDF membrane (Amersham), blocked in non-fat milk, treated with primary antibody and secondary antibodies. Antibody binding was then detected using an ECL chemiluminescence kit. The following antibodies were used: glucocerebrosidase (ab55080 Abcam), actin (AC15 Abcam), LIMP2 (ab106519 Abcam), LC-3 (Cell signalling), cathepsin D (ab40697 Abcam), anti-haemagglutin (covance HA.11) and p62 (ab56416 Abcam). For each antibody, three controls were run alongside the patient samples on each gel. The band intensity for the three controls was averaged and the patient results were expressed as a percentage of the mean control value.