Pd0325901 compound

Treatment with the PD0325901 compound (Selleck) was performed on WT or Shank3B^+/− mice on the C57BL6/J background. The compound was solubilized in 5% NMP, 5% solutol, HS-15, 90% saline with a final concentration of 1 mg/ml. The carrier solution (for the control group) and the solubilized drug (for the treatment group) were aliquoted and stored at −80°C. All experimenters were blinded to the drugs. Aliquots were thawed in a 37°C bath prior to the AM injection and disposed of at the end of the day. Mice in the treatment group received 5mg/kg drug BID 6 days per week. Injection volumes ranged from 70 to 140 microliters and were adjusted on a weekly basis according to individual weights. After 4 weeks of treatment, the cortex was harvested within 4 hours of the last injection and snap frozen in liquid nitrogen. For experiments in which only the whole lysate was needed, samples were homogenized in 50 mM Tris pH 7.5, 0.5% NP-40, 150 mM NaCl, 1 mM EDTA supplemented with protease and phosphatase inhibitors (GenDEPOT). For experiments in which both the whole lysate and the P2 fraction were needed, samples were prepared as described ¹⁹ , then the whole homogenate was solubilized by adding NP-40 to 0.5% final concentration. Lysates were then normalized based on total protein levels and prepared in LDS sample buffer supplemented with reducing sample buffer (Invitrogen).