Anti rip1

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.

Protein isolation and Western Blot were conducted as previously described^{26 ,27 (link)}. In brief, GCs were lysed after 1 to 5 days of culture using RIPA buffer containing protease and phosphatase inhibitors (PI, Thermo Fisher Scientific, Waltham, USA). A total of 10 µg protein per lane was loaded on a 12 % SDS gel and run under constant current (30 mA/gel). After blotting (100 V, 65 min) and blocking with 5 % non-fat dry milk in Tris-buffered saline with Tween 20 (TBS-T, 50 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween 20, pH 7.4), anti-pRIP1(S166), anti-pRIP3(S227) both from Cell Signaling Technology (Danvers, MA, USA) and anti-pMLKL(T357/S358) antibodies were administered to decorate these phosphorylated proteins. To visualize specific binding, HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) was used. As loading controls, anti-RIP1, anti-RIP3 (both from Cell Signaling Technology, Danvers, MA, USA), anti-MLKL (ab184718, Abcam, Cambridge, UK) and anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA) antibodies were used. Preabsorption of anti-pMLKL was previously published^{27 (link)}. All experiments were carried out five times if not described otherwise.