Fusion fx7 detection device

3 to 5 µg RNAs were separated on a 1.5% agarose gel and transferred overnight by capillarity on a nylon membrane (GE Healthcare, #RPN203B) in SSC 10X Buffer. RNAs were then UV-crosslinked to the membrane using a Stratalinker apparatus. Generation and incubation of the membrane with RNA dig-labeled probes was performed using the Dig-Northern-Starter Kit (Roche, #12039672910), following manufacturer’s instructions. Membranes were washed twice in 2X SSC 0.1% SDS and once in 1X SSC 0.1% SDS, for 10 min at 65 °C. Revelation was done according to the kit instructions using a Vilber Lourmat Fusion Fx7 detection device. Oligonucleotides used to generate DNA templates for RNA probes are listed in Supplementary Table 5.

Either whole-cell protein extracts or fractionated cell extracts were separated by SDS-PAGE (10%) and subsequently used for western blotting. Immunoblot analysis was performed using primary antibodies targeting hexokinase II (1:500 dilution; #2106); hexokinase I (1:1000; #2804); N-Myc (1:1000; #9405); c-Myc (1:1000; #9402); PHGDH (1:1000, #13428, all from Cell Signaling Technology). To ensure equivalent protein loading, blots were stained with a primary antibody raised against β-actin (1:2000 dilution; #A5441, Sigma Aldrich Merck, Munich, Germany). Detection and visualization was achieved using HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (GE Healthcare) and the ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, #RPN2236), respectively. Data were analyzed using a FusionFX7 detection device (Vilber Lourmat).