Supplementary Figure Legends
Figure S1. Lack of effects of social isolation on insulin-degrading enzyme and neprilysin in the APP/PS1 mice.
Whole cell lysates were prepared from hippocampi of group and isolated APP/PS1 mice. Insulin- degrading enzyme (n=6) and neprilysin (n=7) were quantified by Western blot analysis.
Figure S2. Fear conditioning during training. The mice received 4 tone-shock pairings, freezing response were no significant difference in pre-training freezing and freezing levels during the 4 training trail among 4 different groups (n=15 in each group).
Supplementary Materials and Methods
-, -, and -secretase activity
-secretase, -secretase, and -secretase activity were measured using commercially available kits (R & D Systems, Minneapolis, MN). Hippocampal tissues were homogenized in supplied buffers. Lystaes were incubated on ice for at least 10 minutes, centrifuged at 10,000×g for 1 minute. Supernatants were collected and protein (200 g) was added to secretase–specific APP peptide substrate (YEVHHQKLV for -secretase activity assay, REEVNLDAEFKR for - secretase activity assay and GVVIATVIV for -secretase activity assay) conjugated EDANS and
DABCYL as the reporter molecules. Cleavage of the substrate by the secretase separates the
EDANS and DABCYL allowing for the release of a fluorescent signal. This signal was read at an excitation/emission wavelength of 355/510 nm. All control and samples were run in triplicates.
Cdk5 Kinase assay
The lysates (400 g) were incubated with Cdk5 antibody (2 g; Santa Cruz) in homogenizing buffer for 1 hr at 4°C, and then with protein G-coupled agarose beads (15 l, Sigma) for 3 hr at
4°C. After centrifugation, the agarose beads washed 3 times using homogenizing buffer. For the
Cdk5 kinase assays, the precipitate was incubated for 30 min at 30°C in kinase reaction buffer
[25 mM Tris (pH 7.5), 10 mM MgCl2, 100 M ATP] containing 0.1 mM histone H1 peptide
(PKTPKKAKKL; Promega) in a final volume of 50L. After the kinase reactions were complete, samples were added to an equivalent amount (1:1) of Kinase-Glo Plus reagent and the mixtures were transferred to a white-well plate. Assay from Promega was according to the manufacturer’s instructions. Luminescence was read on a WALLAC VICTOR-2 plate reader.