<p><b>Panel A:</b> Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) <b>Panel B:</b> Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. <b>Panel C:</b> Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.</p
