FIG. 5.
Effects of SECp43 expression on selenoprotein synthesis and selenoprotein mRNA levels. (A) HEK-293 cells were transfected with zebra fish selenoprotein Pa expression plasmid (Zf selP) in the absence (0) or presence (+) of SECp43 plasmid. Triplicate samples for each condition were labeled with 75Se and the media analyzed by SDS-PAGE and autoradiography. (B) Transfections were carried out as described for panel A, in the presence of SBP2 plasmid, followed by 75Se labeling, SDS-PAGE, and autoradiography. 75Se Zf selP, zebra fish selenoprotein Pa expression plasmid; UGA term, UGA-mediated termination. (C) Transfections of SECp43 plasmid were carried out and mRNA isolated two days after transfection. mRNAs for SECp43 and the indicated selenoproteins were quantitated by hybridization to oligonucleotides spotted on nylon membrane arrays as described in Materials and Methods. TR1, thioredoxin reductase 1; Sep 15, a 15-kDa selenoprotein; D1, deiodinose 1; *, P ≤ 0.05; **, P ≤ 0.01. (D) Cotransfections of HEK-293 cells with Sec-tRNA[Ser]Sec and SBP2-GFP with or without SECp43 plasmid and with one of two GPX expression plasmids. The mRNA levels of the transfected mouse GPX1 constructs with (GPX + intron) or without (GPX − intron) a genomic intron downstream of the UGA codon were quantitated by real-time quantitative PCR analysis using primers specific for the FLAG tag and GPX1 (arrowheads) or unique GPX1 (arrows) sequences. Hypoxanthine phosphoribosyltransferase (HPRT) mRNA levels were quantitated as an internal control for the cDNA synthesis reactions. **, P ≤ 0.01; no symbol, P ≥ 0.05.