A. Western blot analysis of the milk proteins α-casein, β-casein and Wap in the mammary glands of Elf5^+/+ and Elf5^+/− mice. B. Lactalbumin (Lalba) expression in the mammary glands of Elf5^+/+ and Elf5^+/− mice measured by microarray analysis. Expression is shown relative to the virgin Elf5^+/+ sample.

Expression profiles of the 23 genes that consistently clustered with Wap using 2 clustering algorithms, in the mammary glands of Elf5^+/+ and Elf5^+/− mice. Gene expression is shown relative to expression in the virgin Elf5^+/+ condition.

A. The effect of ELF5 expression on MUC4 promoter activity was tested using MUC4 promoter-luciferase reporter gene assays with different regions of the MUC4 promoter. Cells were transfected with either the promoter construct alone or with the promoter construct plus an ELF5 expression vector at molar ratios of 1∶1 or 1∶0.5 (promoter-luciferase vector:ELF5 expression vector). Luciferase expression is shown relative to the expression of the pGL3 promoterless vector. *** p<0.001; **p<0.01. n = 3. B. EMSA was used to determine whether Elf5 could bind the −216 Ets site within the MUC4 promoter. Binding was observed (arrow) in lane 1 when a nuclear extract from a 16.5dpc Elf5^+/+ mammary gland was incubated with a radioactively labelled oligonucleotide encompassing the −216 Ets site from the MUC4 promoter (wt). The binding observed was not evident when the Ets site in the probe was mutated (mt) (lane 2), indicating that the upper band represents specific binding to the Ets sequence. Binding to the wt probe was competed off with a 100-fold molar excess of unlabelled wt oligo (lane 3) but not with mt oligo (lane 4). Binding to the wt probe was not shifted with the addition of anti-ELF5 antibody (lane 5). C. The EMSA from (A) was performed again using a Δ33 ELF5 recombinant protein in place of the nuclear extract. The truncated recombinant ELF5 protein bound the labelled wt oligo (lane 1), but not the mt oligo (lane 2). Binding to the wt oligo could not be competed off with the mt oligo (lane 3), but was successfully competed off using an excess of unlabelled wt oligo (lane 4).

A. A condition dendrogram was generated within the Genespring software suite using an average linking clustering algorithm and a Spearman correlation similarity measure. Individual genes are represented by horizontal bars, which are coloured according to their expression level relative to the virgin Elf5^+/+ (V+/+) sample. B. Principal components analysis was performed in Genespring on all 10 conditions examined by microarray. Conditions are plotted on a 3-dimensional scatter plot according to their variance from the first 3 components. The conditions were normalised to the V+/+ condition.

A. Elf5 mRNA expression in in the mammary glands of Elf5^+/+ and Elf5^+/− mice measured by qRT-PCR. Expression is shown relative to the average expression of Elf5 in the Elf5^+/+ virgin samples. ***p<0.001. n = 3. B. Western blot analysis of Elf5 protein expression in the mammary glands of Elf5^+/+ and Elf5^+/− mice during pregnancy.

A. Muc4 mRNA expression in the mammary glands of Elf5^+/+ and Elf5^+/− mice measured by microarray analysis. Expression is shown relative to the virgin Elf5^+/+ condition. B. Muc4 expression in the mammary glands of Elf5^+/+ and Elf5^+/− mice measured by qRT-PCR. Expression is shown relative to the average expression of Muc4 in the virgin Elf5^+/+ samples. ***p<0.001; *p<0.05. n = 3. C. Western blot examining Muc4 protein expression in Elf5^+/+ and Elf5^+/− mammary glands.