Shank3

Whole lysates of the mouse brain were prepared as described previously (Han et al., 2009 (link), 2015 (link)). Briefly, the striatum and hippocampus of 12-week-old mice were homogenized in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) with freshly added protease and phosphatase inhibitors (Roche). Protein concentration was measured using Bradford Protein Assay (Bio-Rad). Brain lysates were heated in 1x NuPAGE LDS sample buffer (Invitrogen) containing a 1x NuPAGE reducing agent (Invitrogen). From each sample, 10~20 μg of proteins were loaded for Western blotting. Immunoprecipitation (IP) was performed as described previously (Han et al., 2013b (link); Lee Y. et al., 2017 (link)). The GFP-Trap beads (ChromoTek) were used to pull down EGFP-Shank3 proteins and their interactors. The antibodies used for Western blotting were Gapdh (Cell Signaling, #2118), GFP (NeuroMAb, #75-131), Homer1b/c (Santa Cruz, sc-20807), Rhes (Millipore, ABN31), Shank3 (Santa Cruz Biotechnology, sc-30193), phospho-mTOR (S2448, Cell Signaling, #2971), mTOR (Cell Signaling, #2983), and WAVE1 (NeuroMab, 75-048). Western blot images were acquired by ChemiDoc Touch Imaging System (Bio-Rad) and quantified using ImageJ software.