Mem per eukaryotic protein extraction reagent kit

Protein was extracted from P14 retinas from Dscam mutants and wild-type littermate controls using mem-PER Eukaryotic Protein Extraction Reagent Kit (Thermo Scientific, Rockford, IL, USA); 20 μg total protein was loaded into each lane and was separated using 10% acrylamide gel. Blots were transferred to polyvinylidene difluoride (PVDF) membranes (162-0174; Bio-Rad Laboratories, Hercules, CA, USA) and blocked in tris-buffered saline and tween 20 (TBST) (0.05% Tween) and 3% nonfat dry milk. Blots were probed with rabbit anti-VEGF antibody (1:1000; Santa Cruz Biotechnology) for 75 minutes at RT followed by four 5-minute washes in TBST at RT. Anti-VEGF was detected using goat anti-rabbit horseradish peroxidase (HRP)-linked antibody (7074, 1:25,000; Cell Signaling Technology), diluted in 3% milk. Blots were then washed four times for 5 minutes in TBST at RT. Immoblin Western Chemiluminescent HRP Substrate Kit (Millipore) was used to detect antibodies. Blots were then striped and re-probed with rabbit anti-Glyceraldehyde-3-Phosphate Dehydrogenase (anti-GAPDH) antibody (247002, 1:1000; Synaptic Systems, Goettingen, Germany) and detected with anti-rabbit HRP linked antibody (Cell Signaling Technology). Steps for anti-GAPDH were performed identical to anti-VEGF. Densitometry was performed using FIJI to calculate the relative densities between anti-VEGF and anti-GAPDH.