Western blot analysis

LNCaP (3.5 × 10⁵) and 22Rv1 (5 × 10⁵) cells were seeded in 60-mm dishes, allowed to attach overnight, and then treated with DMSO or desired concentrations of PIC for 8, 16, or 24 hours. Cell lysates were prepared in ice-cold lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 1% Triton X-100, 0.1% sodium dodecyl sulfate, 150 mmol/L NaCl, and protease and phosphatase inhibitor cocktail mixture.

The lysates were separated by centrifugation at 14,000 rpm for 15 minutes, and protein content in supernatant fraction was quantified by the Bradford method. Lysates containing 20-30 µg protein were subjected to SDS PAGE, and the proteins were transferred onto polyvinylidene fluoride membrane. After blocking with non-fat dry milk (5%) in TBS containing Tween-20, membranes were probed with a desired primary antibody followed by the appropriate peroxidase-conjugated secondary antibody. The immunoreactive bands were visualized by enhanced chemiluminescence detection system according to the manufacturer’s instructions. To ensure equal protein loading, each membrane was stripped and re-probed with anti-β-actin or anti-GAPDH antibody. The software UN-SCAN-IT5.1 (Silk Scientific, Orem, UT, USA) was used to quantify the protein level changes relative to control.