Grip1

GRIP1 levels in the prefrontal cortex were measured using a western blot, as described in Briand et al., 2014 (link). Briefly, animals were decapitated, and the prefrontal cortex dissected using a brain block (Braintree Scientific). Protein quantification was performed using a Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 µg) were loaded into each well of a Tris-glycine gel (Lonza) and transferred to nitrocellulose membranes (Immobilon). Membranes were blocked with Li-Cor blocking buffer and allowed to incubate in primary antibody solution (GRIP1, 1:2000 (BD Biosciences) and GAPDH, 1:5000 (Cell Signaling)) for 24 hours at 4°C. Membranes were then incubated with fl uorescent secondary antibodies (1:20,000; IR-dye 680 or IR-dye 800, Li-Cor) and imaged on an Odyssey fluorescent scanner (Li-Cor). Western blots were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the percent knockout calculated as a fraction of the average of the GRIP1 levels in GFP-infused mice.