Tween 20

Cells were seeded in a 6-well plate and cultured in a 37 °C, 5.0% CO₂ incubator to adhere to the cells. The old culture medium was discarded and replaced with a fresh medium containing 50 μM of the iron complex. Incubation was then continued for 24 h. After treatment was completed, the cells were washed with PBS. The total protein was extracted using an RNA/protein extraction kit (MACHEREY-NAGEL) according to the manufacturer's instructions. Lysates (50 μg) were subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and then proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Primary antibodies diluted 1% skim milk in PBS with 1.0% Tween-20 (Sigma) and then incubated at 4 °C overnight. Primary antibody: caspase-9 (cell signaling technology (CST), 9508), cleaved caspase-9 (CST, 7237), caspase-3 (CST, 9662), cleaved caspase-3 (CST, 9661), PARP (CST, 9542), cleaved PARP1 (CST, 5625), β-actin (bethyl laboratories, A300-491A). The horseradish peroxidase-conjugated secondary antibodies in 2.0% skim milk in PBS with 1.0% Tween-20 were incubated at room temperature for 1 h. Signals were induced using the chemiluminescent detection reagent (ATTO) and detected using an Amersham Imager 600 (GE Healthcare).

Before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), all samples were mixed with sample buffer containing β-mercaptoethanol (38 (link)) and boiled at 95 °C for 5 min. Poly(vinylidene difluoride) membrane was treated with Tris-buffered saline (TBS)-0.1% Tween 20 (Nacalai Tesque) containing 10% skim milk for 1 h, followed by the primary antibody (nondilution [SOF1], 1:1,000 [SLC2A3, IZUMO1, ACTB, FLAG, and BASIGIN], 1:3,000 [1D4]) for 3 h at room temperature or overnight at 4 °C. After washing with TBS-0.1% Tween 20, the membrane was further probed with the secondary antibody (1:1,000 [SOF1, SLC2A3, IZUMO1, ACTB, FLAG and 1D4], 1:5,000 [BASIGIN]) for 1 h. The protein bands were visualized by Amersham ECL Prime Western blot detection reagent (GE Healthcare).