Protease inhibitor cocktail set 1

Rats were decapitated and bilateral NAc tissue (primarily core) was rapidly dissected from a 2 mm coronal slice prepared with a brain matrix (ASI Instruments). Immediately following dissection, synaptoneurosomes were prepared according to published protocols (Most et al., 2015 (link); Workman et al., 2015 (link); Werner et al., 2018 (link)). NAc punches were homogenized in 500 μl of homogenization buffer [HB; 20 mm HEPES, 0.5 mm EGTA, 1× Proteasome Inhibitor Cocktail Set 1 (Millipore)]. Homogenates were passed through a 100-μm-pore filter and then through a 5-μm-pore filter (Millipore; both filters were prewashed with HB). After homogenates were passed through each filter, filters were washed with 50 μl of HB, and the washes were added to homogenates to maximize yield. Homogenates were then centrifuged at 14,000 × g for 20 min at 4°C. The pellet, which contains the synaptoneurosomes, was frozen on dry ice, stored at −80°C, and ultimately lysed in lysis buffer [0.605 × g Tris-HCl, 0.25 × g sodium deoxycholate, 0.876 × g NaCl, 1 μg/ml PMSF, 5 ml of 20% SDS, and 1× Protease Inhibitor Cocktail Set 1 (Millipore) in 100 ml of dH₂O] for immunoblotting. NAc synaptoneurosomes prepared from individual rats (10 μg protein/lane) were mixed 1:1 with 2× sample treatment buffer (catalog #161–0737, BIO-RAD) and analyzed by SDS-PAGE and immunoblotting. β-Tubulin was used as a loading control.

The corpus callosa were dissected under a Leica stereo microscope (MZ 6; Leica Biosystems, Buffalo Grove, IL), followed by washes in 1x PBS and lysis in ice-cold RIPA buffer (1% Nonidet P-40, 50 mM Tris pH 7.6, 120 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail set 1 (Millipore, Burlington, MA). The lysates were cleared of insoluble materials by centrifugation at 16,000 x g for 10 min at 4°C. Protein concentration was determined by a Bio-Rad protein assay method (Bio-Rad, Hercules, CA) according to the manufacturer's protocol, and equal amounts of protein were used for SDS–PAGE and western blot analysis. The GTP-Rho pull-down assay was performed as previously described (Luo et al., 2011 (link)); in short, tissues were pulverized on liquid nitrogen, lysed in 300 µl of ice-cold RIPA buffer containing protease inhibitors with a cell disruptor for 10 min and homogenized with a 26 G syringe needle. Equal amounts of total protein were incubated with 60 μg GST-RBD beads (Cytoskeleton, Denver, CO) at 4°C for 90 min. The beads were washed twice with lysis buffer and once with TBS buffer. Bound Rho proteins were eluted by Laemmli sample buffer and detected by western blot using mouse monoclonal anti-RhoA antibody (Cytoskeleton, Denver, CO).

Cells were lysed in RIPA buffer (CW2333S, CWBIO, China) supplemented with protease inhibitor cocktail set 1 (539131, Millipore, USA) and phosphatase inhibitor. Protein concentrations were measured using BCA protein assay kits (CW0014S, CWBIO). Equal amounts of total cell lysate were separated using 10% SDS-PAEG (P0014B, CWBIO) and transferred to PVDF membranes (ISEQ00010, Millipore, USA) blocked with 5% bovine serum albumin (0332, Ameresco) for 1 h at room temperature. Then the blots were incubated with the primary antibodies against SHMT2 (1:1,000, GTX125939, GeneTex), E-cadherin (1:1,000, 24E10, Cell Signaling), ZO1 (1:1000, D7D12, Cell Signaling), N-cadherin (1:1,000, D4R1H, Cell Signaling), Vimentin (1:1,000,D21H3,Cell Signaling, β-catenin (1:1,000, D10A8, Cell Signaling), p21^Cip1 (1:1000, 12D1, Cell Signaling), p27^Kip1 (1:1000, D69C12, Cell Signaling), Cyclin D1(1:1000, E3P5S, Cell Signaling), CDK4(1:1000, D9G3E, Cell Signaling), β-actin (1:1000,13E5, Cell Signaling), and GAPAH (1:1,000,D16H11, Cell Signaling) at 4 ℃ overnight. Membranes were incubated in HRP-conjugated secondary antibody (1:2000, 7074S, Cell Signaling) for 1 h at room temperature. The protein bands were exposed using a chemiluminescent substrate (WBKLS0500, Millipore, USA), and quantitatively analyzed using ImageJ [19 (link)].