Journal: Scientific reports

Article Title: Selenocysteine insertion sequence binding protein 2 (Sbp2) in the sex-specific regulation of selenoprotein gene expression in mouse pancreatic islets.

doi: 10.1038/s41598-020-75595-4

Figure Lengend Snippet: Figure1. Sbp2 gene targeting and characterization in Sbp2 βCKO mice. (a) Graphical representation of genetic combinations used to breed Sbp2 βCKO mice. The mouse Sbp2 gene contains 17 exons. Sbp2 βCKO mice are hemizygous for the Mip-Cre-ERT transgene and homozygous for the floxed Sbp2 gene (exon 14 flanked by LoxP sites). Specific expression of Cre-recombinase in β-cells is induced upon tamoxifen binding, which triggers LoxP recombination and excision of exon 14. The Frt site that remained after Flp-induced excision of the NeoR cassette during the making of Sbp2 floxed mice is also excised along with exon 14 upon Cre-Lox recombination in the Sbp2 βCKO mice as shown. Sbp2 qPCR primer-binding locations within exon 14 are also shown. Male (b) and female (d) Sbp2 βCKO mouse islets, liver, and kidney were analyzed for Sbp2 mRNA expression by qPCR and compared to control Sbp2 fl/fl mice lacking the Mip-Cre-ERT transgene (Sbp2 fl/fl) but also treated with tamoxifen. (c) Representative immunoblots showing Sbp2 protein expression for islets, liver, and kidney in Sbp2 βCKO and Sbp2 fl/fl controls in both male (top panels) and female (bottom panels) mice; lanes 1 and 2 are from control mice (Sbp2 fl/fl), and lanes 3 and 4 are from Sbp2 βCKO mice; β-actin was used as the protein loading control. Corresponding quantitation of protein levels was normalized to β-actin expression in arbitrary units and is shown at the right. Between group differences were analyzed by unpaired, two-tailed Student’s t test. N = 6 mice per group; p < 0.05 was considered statistically significant with individual p values as shown.

Article Snippet: Primary antibodies against Sbp2, Gpx3, Dio1, and Txnrd2 (all from Proteintech) as well as Selenop (Santa Cruz) were used to quantify select selenoprotein expression by immunoblotting after separation by SDS-PAGE; β-actin (Cell Signaling) was used as the internal protein loading control.

Techniques: Expressing, Binding Assay, Control, Western Blot, Quantitation Assay, Two Tailed Test

Journal: Scientific reports

Article Title: Selenocysteine insertion sequence binding protein 2 (Sbp2) in the sex-specific regulation of selenoprotein gene expression in mouse pancreatic islets.

doi: 10.1038/s41598-020-75595-4

Figure Lengend Snippet: Figure 2. Selenoprotein gene expression analysis in Sbp2 fl/fl control male and female mouse islets. Quantitative real time-PCR analysis (qPCR) of selenoprotein mRNA abundance in male and female control mice (Sbp2 fl/fl) is shown. Individual selenoprotein mRNA expression is displayed compared relative to the expression of Gpx3, which was the most abundantly expressed selenoprotein mRNA in both male and female islets. Expression was normalized to the housekeeping gene Hprt. Genes susceptible to NMD are marked with the following symbol: ‡. These are selenoprotein pre-mRNAs with a UGA stop codon at least 50–55 nucleotides upstream of an intron. Between group differences were analyzed by unpaired, two-tailed Student’s t test. N = 6 mice per group; p < 0.05 was considered statistically significant with individual p values as shown.

Article Snippet: Primary antibodies against Sbp2, Gpx3, Dio1, and Txnrd2 (all from Proteintech) as well as Selenop (Santa Cruz) were used to quantify select selenoprotein expression by immunoblotting after separation by SDS-PAGE; β-actin (Cell Signaling) was used as the internal protein loading control.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

Journal: Scientific reports

Article Title: Selenocysteine insertion sequence binding protein 2 (Sbp2) in the sex-specific regulation of selenoprotein gene expression in mouse pancreatic islets.

doi: 10.1038/s41598-020-75595-4

Figure Lengend Snippet: Figure 3. Selenoprotein gene expression analysis in Sbp2-deficient male and female mouse islets. Quantitative real time-PCR analysis (qPCR) of selenoprotein mRNA in male (a) and female (b) Sbp2 βCKO islets compared with that of control mouse islets (Sbp2 fl/fl). Individual selenoprotein mRNA expression is displayed compared relative to the expression of Gpx3. Expression was normalized to the housekeeping gene Hprt. Genes susceptible to NMD are marked with the following symbol: ‡. Representative immunoblots showing Gpx3, Selenop, Dio1, and Txnrd2 protein expression in male (c) or female (d) islets of Sbp2 βCKO and control (Sbp2 fl/fl) mice are shown; lanes 1 and 2 are representative islets from control mice (Sbp2 fl/fl), and lanes 3 and 4 are representative islets from Sbp2 βCKO islets; β-actin was used as the protein loading control. Corresponding quantification expressed in arbitrary units is shown at the right normalized to β-actin expression. (See also Supplementary Information 1.) Gene expression of the Selenop receptors Lrp1 and Lrp8 in male and female Sbp2 βCKO islets compared with that of control mouse islets (Sbp2 fl/fl) is shown in (e). Between group differences were analyzed by unpaired, two-tailed Student’s t test. N = 6 mice per group; p < 0.05 was considered statistically significant with individual p values as shown.

Article Snippet: Primary antibodies against Sbp2, Gpx3, Dio1, and Txnrd2 (all from Proteintech) as well as Selenop (Santa Cruz) were used to quantify select selenoprotein expression by immunoblotting after separation by SDS-PAGE; β-actin (Cell Signaling) was used as the internal protein loading control.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Expressing, Western Blot, Two Tailed Test