How do you induce HIF-1 alpha in samples?

Following is a protocol for cobalt chloride induction of HIF-1 alpha: 1. Grow cells on a 15cm dish - confluency 2. Incubate cells with 100 uM CoCl2 for 6 hours (density does not seem to be an issue for induction). 3. Lyse cells in RIPA containing inhibitors for 30-60 minutes. 4. Spin cells in microfuge (4C) on maximum speed for 20 minutes 5. Collect the supernatant (this is the working extract) * The mobility of HIF-1 alpha induced by desferrioxamine or cobalt chloride treatment differs from the mobility of the hypoxia-induced protein. The reason is not known.