Nicotine Exposure Alters Hippocampal and Cortical Proteins

Rats exposed to nicotine (16–21 days) or saline were anesthetized using 5% isoflurane, decapitated, and the hippocampal and cortical tissues were collected, flash frozen, and stored at −80 °C. At the time of immunoblotting, hippocampal and cortical tissues were homogenized, protein content was analyzed, and proteins were separated by 12% SDS-PAGE as described [47 (link)]. Proteins were transferred to Immobilon-P (Millipore, Burlington, MA, USA) membrane and incubated with primary antibodies against rabbit polyclonal anti-ER-β (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), IL-1β (1:1000; Cell Signaling, Danvers, MA, USA), ASC (1:1000; Santa Cruz Biotechnology), Caspase-1 (1:1000; Novus Biologicals, Littleton, CO, USA), and β-Actin (1:5000; Sigma, St. Louis, MO, USA). All data were normalized to β-Actin (monoclonal; 1:1000; Sigma). Immunoblot images were digitized and subjected to densitometric analysis [45 (link)].