Figure 5. Rates of synthesis and degradation of HIF-1α.

A, Rate of synthesis was ascertained from the accumulation of HIF-1α protein over time measured by Western blots, following inhibiton of degradation by the proteasome inhibitor, MG132 in normoxic conditions. Relative abundance of HIF-1α protein was quantified by densitometric analysis of the Western blots. The intensity of the chemiluminescence was measured by a digital imaging system and expressed as relative density units. The protein synthesis inhibitor, cycloheximide (CHX) was used as control. B, Degradation rates were determined by the reduction of HIF-1α protein level at 30 minutes after addition of CHX to cells previously placed in hypoxic conditions for 4 hours. For each concentration of DIM, a control treatment without CHX was used to account for the change in steady state caused by the effect of DIM on the rate of synthesis. C, The relative rate of degradation is calculated as the ratio of the level of HIF-1α protein without CHX over that with CHX for each concentration of DIM and expressed relative to the rate for the DMSO control. The rate of synthesis as measured by the accumulation of HIF-1α at 60 minutes after proteosome inhibition is also shown, relative to that of the DMSO control. Results shown are the average +/− SD of at least three replicates.