Pick1

The Normal-RGCs and LHON-RGCs were lysed in RIPA buffer (Thermo Fisher Scientific). Protein G magnetic beads (Thermo Fisher Scientific) were washed with 0.02% Tween 20 in phosphate-buffered saline (PBS) and then incubated with the antibodies: PICK1 (5 μg; Cell Signaling Technology), Parkin (5 μg; Abcam, Cambridge, MA, USA) and GRIP1 (5 μg; Abcam, Cambridge, MA, USA) for 2 h at 4 °C. The lysate samples containing 500 μg of protein were incubated with antibody-conjugated beads in binding buffer (50 mM Hepes, 125 mM NaCl, 0.1% Triton X-100, 15 μM CaCl₂, 5 μM EDTA) with rotation at 4 °C overnight. The beads were then washed with binding buffer four times followed by the addition of lysis buffer and sample dye. The immunoprecipitates were analyzed by immunoblotting.

The cells were harvested and lysed in RIPA Lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% protease inhibitor. Equal weights of total protein were separated by SDS/PAGE. After the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA), the blots were incubated with blocking buffer (1 X TBST and 5% skim milk) for 1 h at room temperature and then hybridized with the primary antibodies GluR1 (1:1000; Abcam, Cambridge, UK), GluR2 (1:1000; Abcam, Cambridge, MA, USA), PICK1 (1:1000; Cell Signaling Technology, Denver, MA, USA), PKCα (1:1000; Abcam, Cambridge, MA, USA), PINK1 (1:1000; Cell Signaling Technology), Parkin (1:1000; Abcam, Cambridge, MA, USA) and Homer 1b/1c (1:700; Synaptic Systems, Goettingen, Germany) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were obtained by X-ray film exposure.