DEV01651 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. RT-PCR analysis of Eda pathway expression in dermis and epidermis. (A) Epidermis and dermis from dorsal tract skin at day 8, day 9 and day 10 of incubation was manually separated into dermis and epidermis after incubation in 23PBS, 2 mm EGTA. cDNA was prepared from each sample and normalized by levels of Ef1a transcripts. Cek1 [expressed in the dermis (Noramly et al., 1998)] and cytokeratin 17 (expressed in the epidermis, B.A.M., unpublished) were used to  evaluate the purity of these samples. The majority of the Eda expressed is in the dermis, although the level of expression observed in the epidermis appears to exceed that expected from dermal contamination and to confirm that the low-level expression in the epidermis observed in situ is bona fide signal. (B) Amplified bands from the dermal samples analyzed in A were purified and directly sequenced with primers flanking the alternatively spliced region between Eda-A1 and A2. A representative sequence trace is shown with the sequences specific to the A1 and A2 isoforms downstream of the site of divergence indicated above the trace and the six nucleotides inserted into the A1 isoform underlined. The last nucleotide before the point of divergence is indicated by the red arrow, and positions where both the A1 and A2 isoforms share the same nucleotide are indicated by blue arrows. Both upstream of the divergence and at points of shared sequence, a clean trace indicating a single nucleotide at this position is observed. Downstream of the divergence point, traces indicating roughly equal contributions by the nucleotides expected in the A1 and A2 isoforms at these positions are observed. 

• Supplemental Figure 2 -

  Fig. S2. Infection and ectopic Edar expression are confined to the epidermis. Skin from the sample in Fig. 6B was isolated (A) and embedded for cryosectioning (B). Serial sections were taken through this sample and examined. Representative samples from the planes indicated by the yellow (C-E) or red (F) lines in B are shown below. Both Edar and viral transcripts are confined to the epidermis, even in the largest patch of contiguous infection (F). Note also that a few infected cells (e.g. arrow in F) express ectopic Edar (see Fig. 6).