Chemidoc it 510 system

After plasma treatment for 48h, cell protein was extracted using RIPA lysis buffer (Sigma), and the concentration was determined by BCA protein assay kit (Sigma). Protein samples were denatured with 5×protein loading buffer at 95°C for 5min and separated in 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). Subsequently, proteins were transferred to 0.2 μm of polyvinylidene difluoride membranes (Millpore, Billerica, MA, USA) at 70 V for 2.5 h. After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibody and then washed with TBST three times. The horseradish peroxidase (HRP)–conjugated secondary antibodies, goat anti-rabbit IgG and anti-mouse IgG (1:2000) (Abgent, San Diego, CA, USA), were added and incubated for 1h on a horizontal orbital shaker. Images were visualized with ECL chemiluminescent substrate (Millipore, Bedford, MA, USA) using a ChemiDoc-It 510 system (UVP, Upland, CA, USA) and protein bands were analyzed by densitometric analysis using ImageJ software. Primary antibody against human included caspase-3 (1:500), caspase-8 (1:500), caspase-9 (1:500), phospho-p53 (1:200) (Cell Signaling Technology, Danvers, MA, USA), CD95 (1:200) (Santa Cruz, CA, USA) and β-actin (1:1000) (Immuno Way, Newark, DE, USA).

Cell pellets were lysed in lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% Nonidet P40, and 0.25% sodium deoxycholate. Cell debris was removed by centrifugation for 5 min at 14,000 rpm before sample buffer was added. After boiling, samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), which were blocked with PBS containing 5% low-fat milk and 0.1% Tween 20. Membranes were probed with antibodies against human ferritin heavy chain (FTH1) (1:1000) and β-actin (1:1000) (Cell Signaling Technology, Danvers, MA, USA). Membranes were washed with PBS containing 0.1% Tween 20 (PBST) for 30 min and then incubated with HRP-conjugated goat anti-rabbit IgG (1:2000 for FTHI) and anti-mouse IgG (1:2000 for β-actin) for 30 min at room temperature. Membranes were washed in PBST and imaged using a ChemiDoc-It 510 system (UVP, Upland, CA, USA).

Cell pellets were lysed in lysis buffer containing 50 mM Tris,
150 mM NaCl, 1% Nonidet P40, and 0.25% sodium deoxycholate. Cell
debris was removed by centrifugation for 5 min at
4000 × g before sample buffer was
added. After boiling, samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), which were
blocked with PBS containing 5% low-fat milk and 0.1% Tween 20. Membranes were
probed with antibodies against human ferritin heavy chain (FTH1) (1:1000) and
β-actin (1:1000) (Cell Signaling Technology, Danvers, MA,
USA). Membranes were washed with PBS containing 0.1% Tween 20 (PBST) for
30 min and then incubated with horseradish peroxidase-conjugated
goat anti-rabbit IgG (1:2000 for FTHI) and anti-mouse IgG (1:2000 for
β-actin) for 30 min at room temperature. Membranes were
washed in PBST and imaged using a ChemiDoc-It 510 system (UVP, Upland, CA,
USA).