Anti actin

Protein samples from the transfected cells were prepared in 1× SDS gel-loading buffer (Laemmli buffer) without adding β-mercaptoethanol and quantified using the Pierce™ Coomassie Plus Assay kit (Thermo Scientific, Rockford, IL, USA). Without heat denaturation, equal amount of protein samples (50 μg) were loaded on a 10% SDS-polyacrylamide gel. After electrophoresis, the gel was incubated in NBT/BCIP (Roche, Mannheim, Germany) staining solution until the bands corresponding to ALPL were clearly visible. Same protein extracts upon heat denaturation in the presence of β-mercaptoethanol were used for Western blotting. The primary antibody used for the analysis was anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). An anti-actin antibody raised in rabbit anti-actin (Sigma-Aldrich, Saint Louis, MO, USA). The secondary antibody was anti-rabbit HRP-IgG (Cell Signaling Technology, Beverly, MA, USA).

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.