Fig. 1. Expression of p110, p110ß, and p110 in normal and wounded mouse skin. (A) mRNA levels of the PI3K catalytic subunits p110, p110ß, and p110 were assessed by RPA using 10 µg total RNA from murine back skin, tail epidermis, tail dermis and skin-derived primary keratinocytes and fibroblasts or from full-thickness excisional mouse wounds at the indicated time points after injury. Hybridization with a GAPDH-specific riboprobe was performed as a loading control. -p, undigested riboprobes. (B) 30 µg total cellular protein from non-wounded and wounded skin at different time points after injury were analyzed by western blotting for the presence of p110, Ser473-phosphorylated Akt or p85/p55. Coomassie staining of the gel was performed to verify equal loading. High levels of immunoglobulins in early wounds result from invading immune cells. (C) Sections from tail skin and 1- to 14-day full-thickness excisional wounds were stained with an antibody detecting Ser473-phosphorylated Akt. (a) Tail skin showing phosphorylated Akt (red) in the outer root sheath keratinocytes of hair follicles (arrowhead). 1-day wounds harbor high levels of phosphorylated Akt in keratinocytes at the wound rim (arrow, b and higher magnification in c), and in the panniculus carnosus (arrow, b). The eschar (Es) stains non-specifically. (d) Phosphorylated Akt is detected in suprabasal cells of the hyperproliferative epithelium of 5-day wounds (higher magnification in e, arrowheads) but not of 14-day wounds (f). No phosphorylated Akt was detected in normal human epidermis (g), but suprabasal cells were phospho-Akt positive in the hyperproliferative wound epidermis of a 4-day human incisional wound (h, arrow). The basal lamina is indicated by the white dotted line. Nuclei were counterstained with Hoechst 33342 (blue). Bars, 50 m. (D) In situ hybridizations with a digoxygenin-labeled antisense riboprobe (a,b) show p110 mRNA in basal and suprabasal keratinocytes (arrowheads in b) of the normal epidermis and of the hyperproliferative epithelium of 5-day wounds and in dermal fibroblasts (arrows in b). (c) Serial sections incubated with the sense riboprobe revealed only nonspecific background (c). Bars, 100 µm (a); 50 µm (b,c). D, dermis; E, epidermis; Es, eschar; G, granulation tissue; HF, hair follicle, HPE, hyperproliferative epithelium, M, muscle panniculus carnosus.