Purification of connexins and autoradiography: Hearts were perfused as described (see above). 14C-AAP (48 |J.Ci) 10 nmol/L was infused for 15 mins. The infusion was stopped and the heart was prepared according to the method described by Manjunath and colleagues . Briefly, this involved the following steps. Step 1: the hearts were transferred to a solution containing 1 mmol/L NaHCO3, pH adjusted to 8.2 (4°C). Step 2: the superficial fat and large vessels were gently removed. Step 3: the hearts were cut into small pieces. Step 4: phenylmethylsulphonyl-fluoride (PMSF) 1 mmol/L was added. Step 5: after 15 mins gentle stirring at 4°C the hearts were homogenized two times (steps 6 and 7). Step 8: the homogenate was diluted with 1 mmol/L NaHCO3 (pH 8.2), yielding 300 mL of solution, which was filtered through gauze (200 |J.m mesh size). Step 9: the filtrate was centrifuged at 33,000 g for 15 mins, the pellet was dissolved in 1 mmol/L NaHCO3 (pH 8.2) (step 10), and homogenized and centrifuged (33,000 g, 15 mins) (step 11). Step 12: the resulting pellet was resuspended in 100 mL 0.6 mol/L KI and 6 mmol/L Na2S2O3 in 1 mmol/L NaHCO3 (pH 8.2), PMSF was added (step 13) as described for step 4, and the solution was gently stirred overnight (4°C) (step 14). Step 15: the solution was filtered (see step 9), the filtrate was centrifuged (27,000 g, 30 mins) (step 16), the pellet was resuspended in 100 mL 0.6 mol/L KI and 6 mmol/L Na2S2O3 in 1 mmol/L NaHCO3 (pH 8.2) (step 17), homogenized again, and centrifuged (step 18) at 27,000 g for a further 30 mins. The pellet was washed in 70 mL 0.6 mol/L KI and 6 mmol/L Na2S2O3 in 1 mmol/L NaHCO3 (pH 8.2) (step 19) and centrifuged (27,000 g, 15 mins) (step 20). Cheapest online shopping – find Symbicort for Asthma at our place at best fully-licensed pharmacy.