Protein was extracted from cortical tissue by homogenization in RIPA
buffer (Thermo Fisher Scientific) containing phosphatase/protease inhibitor
cocktail (Thermo Fisher Scientific) and centrifuged at 15,000 ×
g for 20 minutes at 4°C before collecting the
supernatant. A 50 μg aliquot of total protein was
separated by electrophoresis in precast 4% to 12% Bis-Tris Gels (Bio-Rad) and
transferred to nitrocellulose membranes. Membranes were blocked in 5% milk
before probing with the following antibodies: rabbit anti-SHIP1 (1:1000, Cell
Signaling, D1163), rabbit anti-β-actin (1:2500, Sigma, A-2066), mouse
anti-PSD95 (clone 6G6-1C9, 1:2500, EMD Millipore, MAB1596), and rabbit
anti-synaptophysin (1:1000, Abcam, ab16659). The secondary antibody used was
peroxidase-labelled anti-rabbit IgG (1:5000, Vector Laboratories). SuperSignal
West Femto ECL (Pierce, #34096) was used to reveal the immunoreactive proteins,
and images were acquired using a Fujifilm ImageReader LAS-4000. Luminescent
immunoreactive protein bands were quantified using Fiji software (ImageJ).
buffer (Thermo Fisher Scientific) containing phosphatase/protease inhibitor
cocktail (Thermo Fisher Scientific) and centrifuged at 15,000 ×
g for 20 minutes at 4°C before collecting the
supernatant. A 50 μg aliquot of total protein was
separated by electrophoresis in precast 4% to 12% Bis-Tris Gels (Bio-Rad) and
transferred to nitrocellulose membranes. Membranes were blocked in 5% milk
before probing with the following antibodies: rabbit anti-SHIP1 (1:1000, Cell
Signaling, D1163), rabbit anti-β-actin (1:2500, Sigma, A-2066), mouse
anti-PSD95 (clone 6G6-1C9, 1:2500, EMD Millipore, MAB1596), and rabbit
anti-synaptophysin (1:1000, Abcam, ab16659). The secondary antibody used was
peroxidase-labelled anti-rabbit IgG (1:5000, Vector Laboratories). SuperSignal
West Femto ECL (Pierce, #34096) was used to reveal the immunoreactive proteins,
and images were acquired using a Fujifilm ImageReader LAS-4000. Luminescent
immunoreactive protein bands were quantified using Fiji software (ImageJ).