Western Blot Analysis of Cell Signaling

SDS-PAGE and Western blot analysis were performed according to the protocol described previously [16 (link)]. Briefly, cells were seeded in a 6-well plate, and after 24 h, treated with or without PEF-III (20 and 30 μg/mL) and incubated till 70–80% confluence. Lysates were prepared using lysis buffer (50 mM TrisCl, pH 7.8, 150 mM NaCl, 1% NP 40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride). The lysates were then resolved on SDS-PAGE and transferred into polyvinylidene difluoride (PVDF) membranes. Next, the membranes were blocked in skim milk (4%) and Tris-buffered solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for 1 h and incubated overnight at 4 °C with specific primary antibodies including anti-human Bcl-2 (Abcam, Tokyo, Japan), Bax (Cell Signaling Technology, Tokyo, Japan), p53 (Cell Signaling Technology), and β-actin (Cell Signaling Technology). After that, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 50 min and then washed with Milli-Q (3–4 times), after which bound antibodies were detected by chemiluminescence reaction using Immuno Star Zeta (Wako) and EZ capture MG (ATTO Corporation, Tokyo, Japan) according to the manufacturers’ protocols.