Figure 3

Expression and activation of Erbb receptors in dorsal epidermis from non-transgenic and K14- Nrg3 transgenic littermates at P7. (A, C, E, G) Skin from non-transgenic littermate. (B, D, F, H) Skin from K14-Nrg3 transgenic founder. (A and B) Erbb1 is expressed on the cell membranes throughout the IFE and ORS. (C and D) Erbb2 is weakly expressed on the cell membranes throughout the IFE and ORS. (E and F) Erbb3 is expressed on the cell membranes throughout the IFE and ORS and is more highly expressed in the granular layers. (G and H) Erbb4 is expressed at very low levels in the IFE and at low levels in the hair follicles of a non-transgenic littermate. Erbb4 is expressed at increased levels in K14- Nrg3 transgenic skin throughout the differentiating layers of the IFE and in the ORS (mainly cytosolic and weak membranous). Basal expression of receptor in the IFE and ORS is denoted by arrows in A-H. The scale bar represents 50 μm. (I) Immunoprecipitation and immunoblot analysis of tyrosine phosphorylation of Erbb1, Erbb2, Erbb3, or Erbb4 immunoprecipitates from dorsal non-transgenic or K14-Nrg3 transgenic skin. Each of the four Erbbs was immunoprecipitated (IP) from 1 mg of lysate and analyzed by immunoblotting (IB) with anti-phosphotyrosine (P-Tyr). The filters were stripped and reprobed with receptor-specific antibodies to determine receptor levels. W denotes wild-type non-transgenic skin sample and T denotes transgenic skin sample.(J) Erbb4 isoforms expressed in the epidermis. JM and CYT Erbb4 isoform levels as determined by semi-quantitative RT-PCR. Dorsal skin displays similar levels of expression of CYT-1 and CYT-2 levels in non-transgenic and K14-Nrg3 transgenic epidermis. Differential expression of JM-a and JM-b levels were observed such that one of the isoforms is preferentially expressed in the K14-Nrg3 transgenic epidermis (T) compared with that isolated from non-transgenic littermates (W). Three independent transgenic lines (T16, T25, T52) were analyzed and compared with three non-transgenic siblings. Gapdh was amplified in parallel as a control.