Figure 2.

HIF-1α protein levels. (A) HIF-1α protein levels of whole cell lysates of mast cells (MCs) (n = 3) after 3 and 24 h under hypoxia and normoxia. HIF-1α was normalized to β-actin (ratio of HIF-1α/β-actin). Significantly more HIF-1α protein was detected after 24 h under hypoxia, not after 3 h. Representative Western blot: total cell extracts (75 μg) were separated by SDS/PAGE (10% gel) and transferred to nitrocellulose membranes. HIF-1α protein (120 kDa) was detected by the primary antibody against HIF-1α (rabbit anti-HIF-1α, Genetex, 2 μg/ml). β-actin (43 kDa) was used as a housekeeping gene (mouse anti-β actin, Santa Cruz). (B) Transcript expression of hif-1α under hypoxia compared to normoxia (n = 3 independent experiments, all PCR runs were performed twice, depicted are the means of each run). MCs were incubated under hypoxia or normoxia for 3 h and incubated with h.i. Staphylococcus aureus Newman/USA 300 at a MOI of 1 for 45 min before RNA was isolated. Data were normalized to the non-regulated housekeeping gene rps9. The x-fold changes of the values from samples incubated under hypoxia were calculated against the normoxic samples. The spontaneous hif-1α gene expression (CTR) as well as infected with h.i. S. aureus Newman/USA 300 under hypoxia compared to normoxia did not change.