Anti atg5 d1g9

Immunoblotting was carried out as described previously^{5 (link)}. Proteins were separated on SDS-PAGE gels, electro-transferred onto polyvinylidene difluoride membranes and incubated overnight at 4 °C with the following antibodies: anti-Atg3, anti-Atg5 (D1G9), anti-Atg7 (D12B11), anti-Beclin (D40C5), anti-caspase-3 (Cell Signaling), anti-Bcl-2 (Millipore), anti-Bax (Millipore), anti-LC3 (Sigma-Aldrich), anti-LXRα (SantaCruz), anti-LXRβ (SantaCruz), anti-Nur77 (Active motif), anti-NOR1 (R&D systems), anti-PARP (Cell Signaling), anti-Vps34 (Cell Signaling), or anti-Actin (Millipore). For visualization, an ECL plus kit (Amersham Biosciences) was used, and chemiluminescence was measured by autoradiography (Supplementary Fig. 9). Specific bands were quantified with ImageQuant software.

Cell lysates were obtained by incubating the samples in TPNE lysis buffer (1x PBS, 300 mM NaCl, 2 mM EDTA, 1% v/v Tritron X-100) supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml protease inhibitor mix (aprotinin, leupeptin, pepstatin A, chymostatin) and 0.4 mM sodium orthovanadate for 20 minutes on ice followed by centrifugation (15 minutes, 20.000 g). Protein concentration was determined with the BCA protein assay kit (Thermo Scientific). For electrophoresis, protein lysates were loaded onto 12% SDS-polyacrylamide gels and blotted onto a PVDF membrane (GE Healthcare). After incubation with anti-Atg5 (D1G9, Cell Signaling Technology), c-FLIP (#3210, Cell Signaling Technology), Mcl-1 (#94296, Cell Signaling Technology) or anti-β-actin (A2228, Sigma-Aldrich) proteins were detected by chemiluminescence (Thermo Scientific).