FIGURE 2

Bmi-1 is expressed in human epidermis. (a) Total RNA (500 ng), prepared from dispase-separated human foreskin epidermis, was amplified by RT-PCR in the presence or absence of 400 nM of each primer, and the resulting DNA product was detected by ethidium bromide staining (in vivo) (arrow). The sample indicated "K" is the RT-PCR product detected using RNA isolated from tAd5-hBmi-1-infected cultured human keratinocytes. M indicates the migration of DNA size markers (nucleotides). (b) Total cell extract was prepared from dispase-separated human foreskin epidermis (in vivo) or from cultured normal human keratinocytes (K) and electrophoresed on an 8% polyacrylamide before immunoblot using anti-Bmi-1 (1:500) and HRP-conjugated sheep anti-mouse IgG secondary antibody (1:5,000). Secondary antibody binding was visualized using enhanced chemiluminescent reagent. -actin levels were measured to normalize protein loading. (c) Human epidermal tissue sections were paraffin embedded, deparaffinized, and hydrated through xylene and a graded alcohol series. Following incubation in 0.3% H₂O₂ for 30 minutes, the tissue sections were washed with PBS for 5 minutes and blocked for 1 hour in PBS containing 1% normal goat serum and 1 mg/ml bovine serum albumin. The primary antibody (mouse monoclonal anti-Bmi-1, diluted 1:50) was added for 2 hours at room temperature followed by incubation for 30 minutes with the secondary antibody (biotinylated horse anti-mouse, 1:1,000) and the signal was visualized using the Vectastain kit (Vector Laboratories, Burlingame, CA). The sections were washed and then counterstained with hematoxylin (Fisher, Pittsburgh, PA) and sealed under coverslips. The top panel (control) was incubated with secondary antibody and the bottom panel (anti-Bmi-1) was incubated with both primary and secondary antibodies. The arrows indicate the epidermal basal layer. Bar=50 m. This experiment was repeated three times with similar results in each experiment.