Figure 3.

A–C, gene expression of edil3 and mfge8 in oviduct regions and four tissues of G. gallus at key events of eggshell biomineralization. Each tissue and stage were determined on six different laying hens (n = 6). A, Edil3 and mfge8 expression in oviduct regions and four tissues of G. gallus. All tissues were harvested at 10 h post-ovulation from 40-week-old hens, except for tibial bone (B), which is from 18-h post-ovulation in 90-week-old hens. B, Edil3 and mfge8 expression in uterus at five key stages of eggshell biomineralization (5–7, 10, and 16 h post-ovulation). C, Edil3 and mfge8 expression in red isthmus at five key stages of eggshell biomineralization (5–7, 10, and 16 h post-ovulation). Relative expression levels of genes were normalized with eight housekeeping genes (see under “Experimental procedures”). Error bars indicate standard deviations. Different letters indicate significant differences in levels of expression (p value <0.05), based on one-way ANOVA and pairwise Tukey test. D, Western blot analysis of EDIL3 and MFGE8 levels in SEM, UF, Ut, and D. For each sample, 15 μg of protein was subjected to electrophoretic separation and electro-transfer. The membranes were probed with anti-EDIL3 (left) or anti-MFGE8 (right) antibodies. AlexaFluor® 680-labeled secondary antibody was used for the detection. Std,: molecular mass standard.