Las 4010 system

Protein samples were fractionated by SDS–PAGE and blotted onto Immobilon-P membranes (Millipore, Burlington, MA, USA) as described previously [50 (link)]. After incubation with the primary antibodies at 4 °C overnight, the blots were detected with the relevant horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Cell Signaling Technology, Danvers, MA, USA) and Western lighting ECL Pro (PerkinElmer, Waltham, MA, USA). Signals were visualized with the LAS-4010 system (GE Healthcare, Chicago, IL, USA) and quantified using the ImageJ software package [51 (link)]. The uncropped images and relative quantification of blots in Figure 1B, Figure 2F, Figure 3E and Figure 6B are shown in Figures S3–S6, respectively.

Cells were treated with 200 μg/ml of ZFE in the presence or absence of 5 μM SP600125 and incubated for 2–24 h at 37°C under a 5% CO₂ atmosphere. The cells were subsequently washed with ice-cold Tris-buffered saline (TBS) and lysed with 100 μl of cell lysis buffer (25 mM Tris-HCl [pH 6.8], 0.8% w/v SDS, 4% w/v glycerol, 0.008% w/v bromophenol blue, 2% v/v 2-mercaptoethanol). The lysate was boiled at 95°C for 5 min, and then centrifuged at 10,000×g for 5 min. Ten microliters of each sample was loaded onto a 12.5% SDS-polyacrylamide gel; proteins were separated by electrophoresis, and then transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) by electroblotting. The membranes were immunoblotted with the following antibodies: anti-LC3 pAb, anti-Atg5 mAb, anti-phospho-JNK pAb, anti-JNK pAb, and anti-β-actin mAb. The bound primary antibodies were incubated with horseradish peroxidase–conjugated antibody against rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and detected using Immobilon Western HRP Substrate detection reagents (Millipore). Band images were acquired using a LAS 4010 system (GE Healthcare Life Sciences, Amersham, UK).

Protein samples were fractionated by SDS–PAGE and blotted onto Immobilon-P membranes (Millipore) as described previously55 . After incubation with the primary antibodies (listed in Supplementary Table 4) at 4 °C overnight, the blots were detected with the relevant horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Cell Signaling Technology) and Western lighting ECL Pro (PerkinElmer). Signals were visualized with the LAS-4010 system (GE Healthcare). Uncropped versions of the blots in Figs 2e and 3f are shown in Supplementary Fig. 15.