FIG. 3.
Expression of HBP1 and Δ393 affects endogenous P47phox mRNA and protein expression. (A) p47 phox mRNA expression is decreased in a stable cell line that constitutively expresses HBP1. RT-PCR for the p47phox gene was performed with 2 μg of total RNA from the control C2C12 muscle cell line and a C2C12 line overexpressing HBP1 (B2 [24]). 18S RNA was used as an internal control, as indicated in Materials and Methods. The reactions were analyzed at the indicated cycle number. MW, molecular weight. (B) Transient expression of HBP1 represses endogenous p47 phox mRNA. 293T cells were cotransfected with 2 μg of F-GFP and 5 μg of HBP1 or control plasmids. The cells were selected by FACS 36 h after transfection. The mRNA was immediately isolated from the GFP-selected cells and analyzed by RT-PCR for the p47phox gene. One microgram of RNA was used with 18S RNA as an internal control with 18S competimers (Ambion; see Materials and Methods). Amplified products were analyzed at the indicated cycle numbers. (C) Transient expression of Δ393 increases p47phox mRNA. Cells were transfected with 2 μg of F-GFP or cotransfected with 5 μg of Δ393. The purification of transfected cells and RT-PCR analysis are as described for panel B. (D) Expression of Δ393 results in increased p47phox protein levels. HEK-293T cells were transfected with either 5 μg of the pmΔ393 (lanes 2 and 3) plasmid or Δ393 (lanes 4 and 5). Western blotting was performed on protein lysates using anti-human p47 phox antisera (a generous gift of Tom Leto) (top). Lane 1 contains untransfected 293T lysate. The blot was stripped and immunoblotted (IB) with anti-HA to show expression of the Δ393 and pmΔ393 proteins.