How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions

Figure 3

Reduced levels of NADPH oxidases 1 and 4 at 18% versus 5% O₂. (a) Representative Western blots showing Nox1 and β-tubulin in PC-3 cells or Nox4 and β-tubulin in C2C12 cells, at 5% and 18% O₂. (b, c) Average Nox1 signal (b) or Nox4 signal (c) standardized to β-tubulin. Total cellular proteins were extracted by treating cells with NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 8.0). Total protein (15 μg per sample) was resolved on 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed for Nox1 or Nox4. β-Tubulin was used as an internal loading and transfer control. All antibodies were purchased from Novus Biologicals; Nox1 (NBP1-31546), Nox4 (NB110-5885), β-tubulin (NB600-936). Data were analysed using two-tailed Student’s t-tests. Bars represent means ± SEM from at least five independent experiments. . The identities of bands at intermediate molecular weight are unknown.

(a)

(b)

(c)