Protein Expression in Irradiated Thyroid

Thyroid samples from 4W and 7M rats at 1, 6, and 12 months after irradiation were analyzed for phospho-p53^Ser15, p16, LC3, and p62 expression. Nonirradiated 4W and 7M thyroid tissues (controls) were removed at the same time and frozen immediately. Total protein was extracted from the tissues^{21 (link),35 (link)}. Proteins (30 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose blotting membranes (GE Healthcare, Tokyo, Japan). Membranes were incubated with anti-p16 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-p53^Ser15 (Cell Signaling Technology, Danvers, MA, USA), anti-LC3, anti-p62/SQSTM1 (MBL), or anti-actin (Sigma-Aldrich, St. Louis, MO, USA) antibodies. This was followed by incubation with an HRP-conjugated anti-mouse IgG antibody (Invitrogen) or HRP-conjugated anti-rabbit IgG (GE Healthcare). Chemiluminescence (ECL Prime, GE Healthcare) was performed according to the manufacturer’s protocol. Protein detection was performed using LAS4000 (FUJIFILM) and quantified using NIH ImageJ software. Data are expressed as previously described^{21 (link)}.