Antibodies used for immunoblots were as follows: anti-ASS1 (Polaris, San Diego, CA, USA), anti-LC3 (Sigma), anti-p62 (Sigma), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling), anti-BCL-2 (Cell Signaling), anti-cIAP1 (Cell Signaling), anti-cleaved PARP (Cell Signaling), anti-caspase 3 (Imgenex, San Diego, CA, USA), anti-RIP3 (Abcam, Cambridge, MA, USA), anti-Atg5 (Santa Cruz, Dallas, TX, USA), anti-Atg7 (Microbiology Laboratories, Woburn, MA, USA), and anti-actin (Sigma). Cells were lysed in RIPA buffer and protein concentrations were determined by BCA kit (Pierce, Waltham, MA, USA). In all, 25–40 μg of proteins was resolved by NuPAGE (Invitrogen) and transferred onto PVDF membranes (Immobilon-P, Millipore, Darmstadt, Germany). Antibody detection was accomplished using enhanced chemiluminescence (Western Lightning, PerkinElmer, Melville, NY, USA).
For co-IP, SK-LMS-1 cells were treated with PBS, 1 μg/ml ADI-PEG20, 20 μM chloroquine, or both for 3 days. Following treatment, cells were lysed in 0.2% NP-40 buffer and protein concentration was determined by BCA kit (Pierce). Lysates were incubated with anti-RIP1 (Cell Signaling) and protein A/G beads (Pierce). The immunoprecipitates were subsequently immunoblotted with anti-RIP3 (Abcam), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling) and anti-actin (Sigma) as described above.
For co-IP, SK-LMS-1 cells were treated with PBS, 1 μg/ml ADI-PEG20, 20 μM chloroquine, or both for 3 days. Following treatment, cells were lysed in 0.2% NP-40 buffer and protein concentration was determined by BCA kit (Pierce). Lysates were incubated with anti-RIP1 (Cell Signaling) and protein A/G beads (Pierce). The immunoprecipitates were subsequently immunoblotted with anti-RIP3 (Abcam), anti-RIP1 (Cell Signaling), anti-caspase 8 (Cell Signaling) and anti-actin (Sigma) as described above.