Figure 2

Expression of HIF-1αFL & HIF-α785 mRNA in human melanoma cells. (A) Schematic representation of the functional domains of both HIF-1α full length and HIF-1α785. Both HIF-1αFL and 785 have various domains in common such as basic helix loop helix (bHLH), Per/Arnt/Sim (PAS), Oxygen Dependent Degradation Domain (ODDD), N-terminal transactivation domain N-TAD, inhibitory domain (ID), nuclear localization signal (NLS) and C-terminal transactivation domain C-TAD. Upon loss of exon 11 in HIF-1α785, part of the ODDD is deleted. This missing region contains the important lysine ⁵³² residue which is acetylated by ARD1 leading to increased stable interaction of HIF-1α with the von Hippel Lindau tumor suppressor. This interaction directs HIF-1α to the ubiquitin-proteasome pathway for degradation under normoxic conditions [5]. (B) Total RNA was extracted from HEMn-LP, SbCl2, WM1366, and WM9. RNA was reverse transcribed using the Advantage RT-for PCR kit^®. Five μL of the resulting cDNA was used in the PCR reaction as described in the Advantage cDNA kit^® manual. Primers (see Methods for sequence) for HIF-1α and HIF-1α785 were designed to specifically amplify each variant with no cross-amplification. Primers for the housekeeping control gene GAPDH were included in the Advantage cDNA kit^®. Control primers amplifying a fragment of the control plasmid included in the Advantage cDNA kit^® were used to ensure optimal PCR conditions.