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. 2019 Nov 8;31:55–66. doi: 10.1016/j.molmet.2019.10.007

Figure 5.

Figure 5

Whole-body ablation of PEMT results in alternative splicing of UCP1 mRNA perinatally. (A) Mitochondrial DNA abundance normalized to nuclear DNA, n = 4. (B) Whole-gel UCP1 immunoblot to detect potentially ubiquitinated UCP1. (C) Abundance of proteins involved in the ER stress response. (D) Amplicon of UCP1 exon 5 in cDNA reverse transcribed from mRNA. (E) Sequence of UCP1 exon 5 in cDNA reverse transcribed from mRNA in the WT and PEMTKO mice, n = 3. (F) Amplicon of UCP1 exon 5 in genomic DNA isolated from the PEMTKO mice. (G) Schematic of alternative 3′ splice junctions in exon 5 of UCP1. (H) mRNA levels of UCP1 splice variants in the PEMTKO mice, n = 4. (I) Measurement of UCP1 protein using commercially available antibodies whose epitopes lie outside the deleted amino acid (AA) sequence in variant D. The antibodies and their epitopes from top to bottom: Alpha Diagnostic UCP11-A, epitope: C-terminus; Abcam ab23841, epitope: AA100-200; Abcam ab10983, epitope: AA145-159; and Cell Signaling 14670S, epitope: C-terminus. All of the antibodies were predicted to detect all of the UCP1 splice variants based on the epitopes. (J) UCP1 protein levels in BAT from the PEMTKO mice at birth. (K) UCP1 protein levels in BAT from 2-week-old PEMT+/- offspring from breeder pairs of either male WT, PEMTKO female, or male PEMTKO, female WT. (L) UCP1 protein levels in BAT from 2-week-old PEMTKO and PEMT+/- mice that were offspring of breeder pairs of either male PEMTKO, female PEMT+/-, or male PEMT+/-, female PEMTKO. Data are expressed as mean ± SEM, *p < 0.05.