Downregulation of HIF1A Expression by HuSH Constructs
HEK293T cells were plated at 2 X 105
per ml (2ml) into a 6-well plate one day before transfection. One (1) ug Hush plasmid was used together with 4 ul TurboFectin 8.0
for transient transfection as suggested by OriGene transfection protocol in the presence of 300 ng SC119189
encoding HIF1A. Seventy two (72) hrs post transfection cells were lysed. Five (5) ug protein were loaded on Invitrogen NuPage Gel system and separated proteins were immunoblotted with anti-HIF1A Rabbit mAb (TA300045
) and reblotted with anti-Erk1/2 (Santa Cruz Biotechnlogy) Abs.