Precooled lysis buffer

HEK293 cells grown in 100 mm dishes were transiently transfected with myc-tagged GGA-wild type (wt), -ΔVHS, -ΔGAT, or-ΔGAE mutants and BACE1-HA. Twenty-four hours after transfection, cells were washed with PBS and lysed in precooled lysis buffer (Miltenyi Biotec) on ice. Cell lysates were centrifuged, and supernatants were transferred to a new tube and incubated with anti-HA-coated MicroBeads (Miltenyi Biotec) for 1 h on ice. Labelled HA-tagged proteins were purified on MicroColumns (Miltenyi Biotec) following the manufacturer’s instructions. Proteins were eluted with 50 μl preheated (95°C) NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) containing 100 mM dithiothreitol and analyzed by LDS-PAGE and Western blotting.

HEK293 cells grown in 100 mm dishes were transiently co-transfected with alphα-synuclein constructs (S1+S2) and empty control vector or myc-tagged GGA3-wild type. Twenty-four hours after transfection, cells were washed with PBS and lysed in precooled lysis buffer (Miltenyi Biotec) on ice. Cell lysates were centrifuged for 15 min at 4°C and 16.000 rcf and supernatants were transferred to a fresh tube and incubated with anti-Myc-coated MicroBeads (Miltenyi Biotec) for 1 h on ice. Labelled Myc-tagged proteins were purified on MicroColumns (Miltenyi Biotec) following the manufacturer's instructions. Proteins were eluted with 50 μl preheated (95°C) NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) containing 100 mM dithiothreitol and analyzed by LDS-PAGE and Western blotting.