Figure 2

Numb5 and Numb6 expression. (A) Western blot detection of Numb5 and Numb6 isoforms from a Numb immunoprecipitation of human and mouse embryonic stem (ES) cells. Another, unknown Numb isoform is indicated with an asterisk. This is confirmed to be Numb from our mass spectrometry analysis and protein mobility on the gel. (B) mRNA analysis of NUMB isoforms in human amniotic fluid (AF) cells by RT-PCR; the AF cells served as a surrogate for human ES cells. Negative control, no template; positive control, NT2/D1 cells after 4 days of retinoic acid treatment; β-actin served as control for the RNA input. (C) Western blot analysis of NUMB5 and NUMB6 in AF cells. Control, the postnatal mouse cortex; loading control, β-actin. (D) Western blot detection of NUMB5 and NUMB6 in breast tumor samples. (E) Real-time PCR analysis of the mRNA extracted from 50 individual mammary tumor samples. The left y-axis and black bar depict the percentage of NUMB5- and NUMB6-positive tumors using the primers specific for each splice variant. The right y-axis and red bar show the ratio of NUMB5 and NUMB6 mRNA to total NUMB mRNA within each sample. (F) Real-time PCR analyses of the NUMB5 and NUMB6 mRNAs extracted from breast tumor specimens in different grades of tumor grades, showing Numb5/6 expression linearly correlated with tumor progression. NT2 cells served as control. Tumor grading was based on Bloom-Richardson system, Error bars indicted standard error of the mean.