Journal: PLOS One
Article Title: Identification of single nucleotide polymorphisms in sheep Mx genes: A premature stop codon abolishes Mx2 protein expression but did not affect fertility and early animal development
doi: 10.1371/journal.pone.0337457
Figure Lengend Snippet: (A) Detection of Mx2 mRNA in wild-type ( MX2 +/+ ) fibroblasts 24 and 48 hours after the stimulation with various IFN-α concentrations using RT-qPCR and the 2-∆∆Ct method with GAPDH expression as a reference. (B) Detection of Mx2 protein in wild-type ( MX2 +/+ ) fibroblasts 48 h after the stimulation with various concentrations of IFN-α using Western blotting (β-actin was stained as a reference). Mx2 and β-actin proteins were labelled using a rabbit polyclonal and a mouse monoclonal antibody, respectively. (C) Interferon-induced expression of Mx2 mRNA in wild-type ( MX2 +/+ ) fibroblasts and fibroblasts heterozygous or homozygous for a SNP (W166*) that terminates the ORF prematurely. Cells were stimulated with 100 IU/mL IFN-α for 48 hours prior to the analysis (Mx2 mRNAs were quantified as described for panel A). Statistical significance: *, p < 0.05; **, p = 0.0027 (adjusted p-values) with Tukey’s HSD by GraphPad Prism (v. 10.5.0). (D) Interferon-induced expression of Mx1 and Mx2 proteins in IFN-stimulated wild-type fibroblasts ( MX2 +/+ ) and fibroblasts heterozygous or homozygous for a SNP (W166*) that terminates the ORF prematurely. Mx1 protein was labelled using a rabbit polyclonal antibody (Mx2 and β-actin were labelled as described for panel B). (E, F) Accumulation and intracellular localisation of Mx1 and Mx2 in wild-type fibroblasts ( MX2 +/+ ) and fibroblasts heterozygous or homozygous for a SNP (W166*) that prematurely terminates the ORF. Cells were either stimulated with 100 IU IFN-α or left untreated, fixed 48 hours after the stimulation, and immunostained using polyclonal antibodies specific for Mx1 (E) or Mx2 (F). To visualize nuclei, cellular DNA was stained using DAPI. Green, Mx-specific staining; blue, DNA staining. Scale bars, 500 µm. All experiments were conducted at least twice.
Article Snippet: Blots were labelled using Mx1- and Mx2-specific rabbit polyclonal antibodies (Cat# PA5–22101 and Cat# PA5–102005, respectively; obtained from Thermo Fisher Scientific); the β-actin-specific rabbit mAb D6A8 (Cat# 8457) was obtained from Cell Signaling Technology (Danvers, MA, USA) and the HRP-conjugated sheep anti-rabbit polyclonal antibody (Cat# ab6795, RRID:AB_955446) was obtained from Abcam (Waltham, MA, USA).
Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining
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