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. 2014 Apr 17;9(4):e95416. doi: 10.1371/journal.pone.0095416

Figure 6. SRA not SRAP transient expression upregulates IR transcription.

Figure 6

A, Schematic presentation of the SRA1 gene and expression constructs used in the reporter assay. The human SRA1 gene contains five exons as indicated by the grey boxes. “SRA Only” contains the human SRA core sequences from exons 2–5 that only express SRA RNA. SRA-WT is the full-length human Sra1 cDNA that expresses both SRAP and SRA mRNA. SRA-RNA is the SRA-WT cDNA in which codon 13 was mutated from ATG to TGA, so that SRAP is not expressed. SRAP-SDM1/7 and SRA-RNA-SDM1/7 are plasmids that are based on the SRA-WT and SRA-RNA constructs with introduction of silent nucleotide mutations in the stem loops 1 and 7, which disrupt the RNA stem loop structures (and hence impair coactivation by SRA [34]) without changing the amino acid sequence of the encoded SRAP. B, SRA upregulates IR transcription in 3T3-L1 cells. 3T3-L1 preadipocytes were cotransfected with either empty vector (pSCT, 500 ng), pSCT-SRA Only (500 ng), or other SRA/SRAP constructs (500 ng) as indicated and IR promoter (pIRP-Gluc, 100 ng in lanes 1–8) or pGluc-Basic (lanes 9–11) driven luciferase expressing plasmids. Forty-eight hours post-transfection, luciferase activity was measured. Data were expressed as fold change after normalization with the total cell protein content of each well. These results are representative of five independent experiments. Error bars indicate the standard deviation. The results of statistical analyses by ANOVA followed Scheffe’s test, comparing bar 1 with bars 2–8, and bar 9 with bars 10–11 and with bars 2 and 4, are shown in the figure (*, p<0.05). C, Confirmation of SRA overexpresion in the transient transfection in B by RT-qPCR, using human SRA primers and normalized to Ppia (cyclophilin A). The data are presented as the mean ± S.D. relative to the SRA expression in pSCT cells set at 1. D, Immunoblot using anti-SRAP indicating SRAP expression in the transient transfection in B (endogenous SRAP expression in lanes 1, 2, 4, 6, and 9–11 was visible only with a longer exposure). Immunoblotting with anti-β-actin served as a loading control.