Hrp conjugated goat anti mouse or anti rabbit secondary antibodies

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).

Either whole-cell protein extracts or concentrated conditioned medium was separated on gradient (4-12%) SDS-PAGE and used for western blotting. Immunoblot analysis was performed using anti-NRG1 (Cat.# AF2015, R&D, Minneapolis, MN, USA), 1:500; anti-phospho-NTRK1 (Cat.# 4621, Cell Signaling, Danvers, MA, USA), 1:1 000; anti-β-actin (Sigma-Aldrich, St Louis, MO, USA), 1:5 000, and HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (GE Healthcare). ImageJ 1.42q (W. Rasband, NIH, Bethesda, MD, USA) was used to quantify signal intensities.