Comprehensive Western and Northern Blot Protocols

For Western blot analyses, cell lysates were separated using 10% SDS-PAGE gels under reducing conditions, transferred to PVDF membranes with the TransBlot turbo system (BioRad) and membranes blocked using fish gelatin (Nordic). Anti-c-Myb (Millipore) and anti-β-actin (Abcam) were used in combination with goat anti-mouse-HRP secondary antibody (Dako), while anti-GLUT1 (Millipore) was used in combination with goat anti-rabbit-HRP (DAKO), and proteins visualised using ECL reagents (Santa Cruz) and a ChemiDoc CCD camera (BioRad). The small RNA Northern blot protocol was adapted from reference (26 (link)). Briefly: 100 - 500 ng total RNA was separated in denaturing urea 15% PAGE gels in TBE buffer before electrophoretic transfer to positively charged nylon membranes (Amersham). RNA was cross-linked to the membrane in a UV chamber (Stratagene), and then blocked using Ultrahyb (Ambion) for 1 hr at 50°C. Double-DIG labeled LNA probe (1 nM, Exiqon) diluted in Ultrahyb was incubated overnight at 50°C, before the membrane was washed in 0.2× SSC buffer, followed by probing using anti-DIG-HRP antibody (Roche) in combination with DIG wash & block buffer kit (Roche), and detection by ECL reagents.