Fig 1: Schematic representation of the MKRN3 gene with various predicted transcription factors. The various transcription factors are indicated with oval shapes. The positions of the mutations identified are indicated in relation with TSS. (A) Wild-type MKRN3 promoter region. (B) MKRN3 promoter region with the novel MKRN3:g.-166G>A mutation. (C) MKRN3 promoter region with the novel MKRN3:g.-865G>A mutation and (D) MKRN3 promoter region with the novel MKRN3:g.-886C>T mutation. TSS: transcription start site; HMX2: H6 Family Homeobox 2; PRDM14: PR/SET Domain 14; SOX4: SRY-Box 4.
Fig 2: MKRN3 5'-UTR in silico predictions. (A,B) Predicted MKRN3 5'-UTR mRNA secondary structures showing the MFE and centroid MFE secondary structure, respectively, for the wild- type and MKRN3:g.+13C>T mutant. (C) Position of the Motif Ten Element (MTE) in relation with the MKRN3:g.+13C>T mutation. Black arrows indicate the position of the MKRN3:g.+13 nucleotide.
Fig 3: DNA sequencing analysis. Part of the sequencing electropherograms of the MKRN3 proximal promoter showing the novel heterozygous mutations identified; rs184950120 (MKRN3:g.+13C>T), rs188505875 (MKRN3:g.-166G>A), rs139233681 (MKRN3:g.-865G>A), and rs74005577 (MKRN3:g.-886C>T). For each mutation the corresponding normal sequencing electropherograms is showed.
Fig 4: (A,B) shRNA knockdown efficiency on (A) RNA and (B) protein Mkrn3 levels. GnRH expressing GN11 cells were treated with the indicated shRNA. (A) Total mRNA analyzed by RT-qPCR. Relative expression of Mkrn3 was analyzed with respect to the 18S gene. (B) Whole cell lysates were analyzed by western blotting with Mkrn3 antibody. The ß-Actin stained membrane serves as loading control. The shMkrn3 reduces the mRNA and protein levels of Mkrn3. As a negative control a shRNA containing a scrambled sequence was used (shScrample). (C) The novel MKRN3 promoter/5'-UTR mutations reduce the promoter activity in GN11 cells. The MKRN3 promoter reporter gene constructs (spanning nucleotides -984_+99 relative to TSS) containing the MKRN3:g.+13C>T, MKRN3:g.-166G>A, MKRN3:g.-865G>A, and MKRN3:g.-886C>T mutations were transiently transfected in GN11 cells. Luciferase activities were calculated relative to the wild-type MKRN3 promoter reporter construct. Results are the average of three independent experiments with each sample assayed in triplicate. *P < 0.0001; **P < 0.001; ***P < 0.05.
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