Protein Extraction and Western Blotting

Whole-cell extracts were obtained from 1.5 × 10⁶ cells using RIPA buffer (Cell Signaling Technologies Inc., Danvers, MA). For cytoplasmic and nuclear protein extraction, the Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) was used, and extraction was carried out according to the manufacturer’s instructions. Western blotting analyses were performed as previously reported [54 (link)], with primary antibodies against β-actin (Sigma-Aldrich), NF-κB (Santa Cruz, Dallas, Texas, USA), α-tubulin, PARP1, phospho-IkBα and phospho-NF-κB (Cell Signaling Technologies). Membranes were scanned and analyzed using the Odyssey^® infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA) and Odyssey 3.0 imaging software. Equal loading and quality of cytosolic and nuclear proteins were determined using α-tubulin and PARP1 expression levels, respectively. The relative protein expressions were normalized using the quantified level of β-actin expression for total lysates, α-tubulin for cytosolic extracts and PARP1 for nuclei extracts, as previously reported [54 (link)]. The data presented are representative of three separate experiments with similar results.