Sk-N-SH cells were incubated at 37&degC for 24h with vehicle control (0 &microM) and 1 &microM of kainic acid (ab120100). Decrease expression of nociceptin receptor in SK-N-SH cells correlates with an increase in kainic acid concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 30&microg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab66219 at 1 &microg/ml and ab8227 at 1 &microg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.