Fresh hippocampus was dissected from the following strains at 2 months-of-age: BXD2, BXD22, BXD28 and BXD42. Extraction of nuclear and soluble protein was performed following a standard protocol (see Supplementary Methods and Li et al.38). Immunoblotting was performed using the WesternDot 625 Goat Anti-Mouse Western Blot Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, 25 μg of total protein (nuclear and soluble fractions combined) was loaded into each well of a 4–12% NuPAGE gel (Invitrogen). Anti-Per3 primary antibody was used at a concentration of 0.2 μg ml−1 and immunopositive bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and the HRP-conjugated secondary antibody provided with the WesternDot kit. Blots were scanned using AlphaImager HP, and the intensity of each immunopositive band was quantified using AlphaView Imaging Software (version 3.0.3.0, AlphaInnotech Corp, Santa Clara, CA, USA). Unpaired t-tests were performed to determine if significant differences in protein levels were present between low (BXD2 and BXD22) and high (BXD28 and BXD42) strains.
