Synaptic fraction H P1 S2 P2 PSD95 CREB H P1 S2 P2 PSD95 CREB Sophie Laguesse date 2015.06.23 experiment number SL46 Aim: Test Prosapip1 expression after synaptic fractionation from Nac of mice after 24 hours of withdrawal Method: Mice experienced intermittent access 20% alcohol 2 bottle choice drinking session during eight weeks. Control animals had access only to water. 24 hours after the last alcohol session, mice were sacrificed and Nac were immediately removed. Nac were homogenated in 250uL cold Krebs buffer+sucrose 0,32M+phosphatase/protease inhibitors using a needle. 60 ul were kept as total homogenate (H) and were lysed by adding 120uL RIPA buffer. The samples were centrifuged 10min at 4 1000g and the supernatant was removed (=S1). 170 ul of Krebs-glucose were added to the pellet and samples were centrifuged a second time at 1000g 10min 4deg. Supernatant was removed and added to S1 (=S1 total). 120ul RIPA buffer was added to the pellet (=P1). S1 total samples were centrifuged 20min 16000g 4deg. Supernatant was kept for quality control (=S2) and 120uL RIPA buffer was added to the pellet (=P2). All P2 fractions were centrifuged 10min 10000g and supernatant were used for protein concentration determination. Prosapip1 and Actin expression were determined by Western Blot. Quality control of synaptic fractionation: PSD95 and CREB expression were determined by WB for one control (C2) and one binge sample (WD1). Water Alcohol (WD) Prosapip1 Actin Synaptic fraction Quality control WD1 sample H P1 S2 P2 PSD95 CREB Prosapip1 in synaptic fraction 200 ** 150 Prosapip1/actin (% of the control) 100 50 Water Alcohol (binge) Quality control C2 sample H P1 S2 P2 PSD95 CREB Conclusion: Prosapip1 expression is significantly increased in the Nac of mice after 24 hours of withdrawal in the isolated crude synaptic fraction. This confirm the increased expression observed in the total homogenate of Nac.