Anti p62

To monitor protein levels, frozen muscles were pulverized by means of Qiagen TissueLyser and protein extracts were prepared in an appropriate buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 10% glycerol, 2% SDS, 1% Triton X-100, Complete EDTA-free protease inhibitor mixture (Roche), 1 mM PMSF, 1 mM NaVO₃, 5 mM NaF, and 3 mM β-glycerophosphate. 40 μg of total proteins was loaded, according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis, in commercial 4-12% acrylamide gels (Thermo Fisher Scientific), and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) by semidry electrophoretic transfer. Blots were blocked for 1 hour at RT with 5% nonfat dry milk (Bio-Rad) in TBS-tween (0.5 M Tris, 1.5 M NaCl, and 0.01% Tween) solution and incubated at 4°C with primary antibodies. Secondary antibodies were incubated 1 hr at RT. The following primary antibodies were used: anti-LC3 (1 : 1000, Cell Signaling), anti-p62 (1 : 5000, Sigma-Aldrich), anti-actin (1 : 20000, Santa Cruz), and anti-MCU (1 : 1000 Sigma-Aldrich). Secondary HRP-conjugated antibodies were purchased from Bio-Rad and used at 1 : 5000 dilution.

For Western blotting, cell lysates in ice-cold RIPA buffer were centrifuged and the supernatants were assayed for protein content. About 20 to 30 μg of proteins were fractionated on 7.5–15% polyacrylamide gel and transferred onto PVDF membrane from Millipore Corporation (Billerica, MA, USA). Nonspecific binding sites were blocked for 1 hour at RT in 20 mM Tris-HCl (pH 7.4) buffer, 55 mM NaCl and 0.1% Tween 20 containing 5% non-fat dry milk (blocking buffer). Membranes were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. Primary antibodies used are anti-LC3, anti-cyclin D1, anti-p62, anti-actin and anti-pERK1,2 antibodies from Sigma Aldrich (St. Louis, USA), anti-p53 and anti-pEGFR antibodies from Santacruz Biotectnology (SantaCruz, CA, USA), anti-cyclin B2 was from Abcam (Cambridge Science Park, Cambridge, UK). All these antibodies are dissolved in blocking solution. After extensive washings and incubation with the respective horseradish peroxidase-labeled secondary antibodies, protein presence was visualized by enhanced chemiluminescence reaction from Pierce Biotechnology (Rockford, IL, USA). Band relative densities obtained using Alliance 4.7 UVITEC (Cambridge, UK) were normalized to actin and values were given as relative units (R.U.).

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.