Identification of 57-kDa PXDNL as hPMR1. (A) Triplicate cultures of U2OS cells were transfected with plasmids expressing GFP (lanes 4–6), xPMR1 (lanes 7–9), or 57-kDa PXDNL (lanes 10–12) together with plasmids expressing albumin (substrate) and luciferase (control) mRNA. Cytoplasmic RNA recovered from each transfectant was analyzed by RNase protection assay. Lanes 2 and 3 contain the probes without (−) or with RNase (+) treatment. (B) Triplicate cultures of tetracycline-inducible MCF-7 cells at 30%–40% confluency were cotransfected with 0.2 μg each of plasmids expressing Xenopus albumin mRNA, luciferase mRNA, and 0.75 μg of pcDNA4/TO-Flag-hPMR1-Tev-biotin (57-kDa PXDNL). The cells were cultured for 18 h without (−Dox) or with (+Dox) doxycycline, and DRB was added at time = 0 to block transcription. Cytoplasmic RNA isolated at 0, 4, and 8 h was analyzed by quantitative RT-PCR, and albumin mRNA was normalized to luciferase mRNA. The mean value at time 0 for albumin normalized to luciferase RNA was arbitrarily set to 1. Shown are the mean ± SD from triplicate cultures; *P < .05, **P < .001 by Student's t-test. The lower panel is a Western blot of cytoplasmic protein recovered from cultures treated ±doxycycline at each time point probed with antibody to the Flag tag on hPMR1.
