JCS03070 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. High levels of Klf5-tg expression compared to the endogenous Klf5 levels in the skin. Skin biopsies were briefly homogenised in lysis buffer (20 ml per 10 mg of tissue). Equal amounts of 2X SDS loading buffer was subsequently added and the samples were kept in a boiling water bath for 5 minutes. Five ml of each sample was resolved on a 4-15% tris Hcl poly-acrylamide gradient gel (Bio rad). Following electrophoresis the proteins were transferred to Immobilon-P transfer membranes (Millipore). Primary antibodies used were- goat anti Klf5 (Santa Cruz) 1:1000; mouse anti-bactin (Sigma) 1:5000. Horseradish peroxidase-conjugated secondary antibodies were used. Protein-antibody interaction was visualised by chemiluminescence using the lightningTM chemiluminescence reagent plus kit (PerkinElmer).

• Supplemental Figure 2 -

  Fig. S2. Verification of the keratinocyte origin of the cells analysed by FACS. To verify the keratinocyte origin of the analysed cells the CD34low a6low, CD34low a6high, a6low and CD34high a6high cell populations were sorted out and 10 000 cells were used for cytospin. Cells were fixed for 30 sec in acetone at room temperature followed by 10 min incubation in methanol at room temperature. Cells were stained with a rabbit polyclonal keratin 5 antibody (1:1000, Covance). Subsequently sections were incubated for 30 minutes with a 1:200 dilution of goat-anti rabbit secondary antibody (Vector Laboratories) and avidin-HRP (horseradish peroxydase) conjugate (Zymed). At least 500 cells were counted in 3 fields. The number of keratin 5-positive cells was 98,5% for the CD34low populations and over 99% for the CD34high populations

• Supplemental Figure 3 -

  Fig. S3. Immunohistochemical analysis of CD34 expression on sections of adult skin antibody, Abcam- 1:50)