*4.4. Immunoblots*

A total of 6 × 10<sup>6</sup> A549 cells were seeded in a 58 cm<sup>2</sup> Petri dish (Celltreat) per condition and allowed to grow for 24 h at 37 ◦C and 5% CO2. A549 cells were then treated with (1) 1 ng/mL ricin, (2) 100 ng/mL cell death ligand/cytokine (TRAIL, TNF-<sup>α</sup>, or FasL), or 3.) 1 ng/mL ricin combined with 100 ng/mL cell death ligand/cytokine for 4 h at 37 ◦C and 5% CO2. Following this, A549 cells were lysed using 1% triton-X-100 (Sigma) with 1× Halt protease inhibitor (Thermo Fisher) in 1× PBS on ice for 30 min. Cells were then sonicated on ice 3× (20 sec pulses) at an output of 10%. Cell lysates were centrifuged at 14000 rpm at 4 ◦C to remove nuclear material. A549 cell lysates were run on SDS-PAGE and transferred to a PVDF membrane and blocked in 1× TBS with 0.1% tween-20 and 5% milk for 30 min at room temperature. The blots were then incubated with diluted primary antibody in 1× TBS with 0.1% tween-20 and 5% milk overnight at 4 ◦C. All primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), unless otherwise indicated. Primary antibodies were used at the following dilutions: anti-human caspase-3 (1:1000), anti-human/mouse caspase-6 (1:1000), anti-human/mouse caspase-7 (1:1000), anti-human caspase-8 (1:1000), anti-human caspase-9 (1:1000), and anti-human GAPDH (1:10,000). After washing with 1× TBS with 0.1% tween-20 and 5% milk, the blots were incubated with secondary HRP-conjugate antibodies (1:5000) for 1 h at room temperature. Blots were developed by chemiluminescence and read in a Bio-Rad ChemiDoc XRS+.
