Digital presenter

Frozen tumors were directly homogenized in PBS-containing protease inhibitors and 1% Triton. After debris removal, protein concentrations were assessed using the BCA assay. Tissue lysates (200 μg) were processed using the Mouse XL Cytokine Array Kit (R&D Systems). The membranes were scanned with a Samsung Digital Presenter with a 720P HD document camera with a 14x optical zoom and 3x digital zoom image and quantified by Image J.

Cell lysates were prepared in ice-cold RIPA buffer, supplemented with a protease inhibitor cocktail. Samples were centrifuged at 10,000g for 5 min at 4°C and supernatants collected. Protein concentrations were determined using the Pierce BCA assay. Laemmli buffer was supplemented with β-mercaptoethanol and incubated for 5 min at 95°C. 5 μg of total proteins was loaded on a 4–12% SDS–PAGE run at 150 V and then transferred to PVDF membranes during 1 h at 100 V. Membranes were saturated in PBS/Tween/5% BSA overnight at 4°C, followed by incubation with either mouse anti-OMA-1, anti-OPA1, anti-DELE1, anti-TOM20, and anti-actin antibodies at 4°C (see Supplemental Data 1). Membranes were washed and incubated with HRP-linked goat anti-rabbit IgG or HRP-linked goat anti-mouse IgG for 1 h at RT. The antigen–antibody complex was detected using ECL, according to the manufacturer’s instructions. Images were captured on autoradiography films and scanned using Samsung Digital Presenter with a 720P HD document camera with a 14× optical zoom and 3× digital zoom. Signals were quantified with ImageJ software and normalized using actin signal intensity as a reference.