Anti homer1

The experimental process is as described in the previous study (19 (link)). PFC and hippocampal tissues of mGluR5^−/− mice and wild-type littermates (group three) were collected and lysed in 100–300 μl of Radio Immunopreciptation Assay (RIPA) lysis buffer (10 mM Tris, 1 mM EDTA, 0.5% NP-40, 150 mM NaCl, and 1% Triton X-100 at pH 7.4). The RIPA lysis buffer contained a 1:100 (v/v) ratio of a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche). The BCA protein assay (Pierce) was used to quantify the total protein samples (20–40 μg), and then the samples were resolved via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The primary antibodies were as follows: anti-β-actin (1:1,000, Cell Signaling Technology); anti-mGluR5 (1:1,000, Abcam); anti-NR2A (1:1,000, Cell Signaling Technology); anti-NR2B (1:1,000, Cell Signaling Technology); anti-PSD95 (1:1,000, Abcam); anti-homer1 (1:1,000, Abcam); and anti-Erk1/2 (1:1,000, Cell Signaling Technology). The enhanced chemiluminescence (ECL) detection method (Advansta) was used to visualize all Western blots. Image J software (version 1.47) was used to quantify the scanned images.

Brains were removed from Shank3B^−/− mice and WT mice and sectioned into 1-mm-thick coronal sections at the end of the study. The tissues of each brain area were extracted from the sections according to the mouse atlas (The Mouse Brain in Stereotaxic Coordinates, second edition, by George Paxinos and Keith B.J. Franklin). The collected tissues were lysed in 100–300 μL of RIPA lysis buffer (10 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, and 1 mM EDTA at pH 7.4) containing a 1:100 (v/v) ratio of a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche). We used the bicinchoninic acid protein assay (Pierce) to quantify total protein samples (20–40 μg). Then, the samples were resolved via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The primary antibodies were as follows: anti-β-actin (1:1,000, Cell Signaling Technology); anti-Shank3 (1:1,000, Abcam); anti-mGluR5 (1:1,000, Abcam); anti-NR2b (1:1,000, Cell Signaling Technology); and anti-homer1 (1:1,000, Abcam). All western blots were visualized using the enhanced chemiluminescence detection method (Advansta). The scanned images were quantified using ImageJ software (version 1.47).