Figure 2. Time- and dose-dependent regulation of NAD(P)H oxidase subunit mRNA expression in response to laminar shear stress.

The mRNA expression of NAD(P)H oxidase subunits gp91^phox and p47^phox was quantified by standard calibrated competitive reverse transcription-polymerase chain reaction (RT-PCR) (A) and normalized to 18S rRNA RT-PCR. The method compares amplification of an gp91^phox and p47^phox cDNA fragment from reverse transcribed total RNA of human endothelial cells (upper lane, longer fragment) versus different concentrations of an internally deleted and reverse transcribed cRNA standard (lower lane, shorter fragment) by PCR. Serial 1 : 3-dilution of appropriate cRNA standard was used. The PCR fragments were separated on agarose gels and stained with ethidium bromide. In time-course studies, human endothelial cells were exposed to laminar shear stress (ss) of 30 dyne cm⁻² for 1, 4, 12 or 24 h and mRNA expression of NAD(P)H oxidase subunits gp91^phox (B) and p47phox (C) was quantified. The dose-dependent regulation of gp91^phox (D) and p47^phox (E) was studied in HUVEC exposed to long-term (24 h) laminar shear stress (ss, 1, 5, 10, 15, 30, 50 dyne cm⁻²). Values are given as means ±s.e.m. as a percentage of control; n ≥ 5 each; *P < 0.05 versus control.