Early Steps of Insulin Action in the Skin of Intact Rats Fabiana F.F. Pelegrinelli, Ana C.P. Thirone, Alessandra L. Gasparetti, Eliana P. Araujo, Lício A. Velloso, Mario J.A. Saad  Journal of Investigative Dermatology  Volume 117, Issue 4, Pages 971-976 (October 2001) DOI: 10.1046/j.0022-202x.2001.01473.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Time course and dose–response of insulin-stimulated tyrosine phosphorylation of IR and IGFR in the skin of intact normal fasted rats. Skin extracts from rats injected with saline or insulin (at the time and dose indicated) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with αIR (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (A). Tissue extracts stimulated by different doses of insulin were immunoprecipitated with αIR (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (B). Tissue extracts stimulated by different doses of insulin were immunoprecipitated with αIGFR (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (C). For controls (last lane in each blot), IgG from normal rabbit serum was used to immuno precipitate skin tissue extracts. These results are representative of five independent experiments. Journal of Investigative Dermatology 2001 117, 971-976DOI: (10.1046/j.0022-202x.2001.01473.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunoperoxidase staining of rat skin fragment using anti-IR polyclonal antibody. Panoramic and detailed views of control staining (primary antibody was a rabbit preimmune serum) (A, C). Panoramic and detailed views of IR-specific staining showing the presence of antigen in cell bodies of the epidermis, hair follicles, and at lower concentration in fibroblasts of the dermis (arrow) (B, D). Journal of Investigative Dermatology 2001 117, 971-976DOI: (10.1046/j.0022-202x.2001.01473.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Time course of insulin-stimulated tyrosine phos phorylation of IRS-1 and IRS-2, and the association of IRS-1 with PI 3-kinase in the skin of intact normal fasted rats. Skin extracts from rats injected with saline (–, 0 min) or insulin (+, 1, 3, and 5 min) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with αIRS-1 (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (A). The same blot was incubated with αPI3K (1 µg per ml) (B). Skin extracts were also immunoprecipitated with αIRS-2 (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (C). For controls (last lane in each blot), IgG from normal rabbit serum was used to immunoprecipitate skin tissue extracts. These results are representative of five independent experiments. Journal of Investigative Dermatology 2001 117, 971-976DOI: (10.1046/j.0022-202x.2001.01473.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Time course and dose–response of insulin-stimulated tyrosine phosphorylation of Shc and association of Shc with Grb2 in the skin of intact normal fasted rats. Skin extracts from rats injected with saline or insulin (at the time and dose indicated) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with αShc (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (A). The same blot was incubated with αGrb2 (1 µg per ml) (B). Tissue extracts stimulated by different doses of insulin were immunoprecipitated with αShc (2 µg per ml) and immunoblotted with αPY (1 µg per ml) (C). For controls (last lane in each blot), IgG from normal rabbit serum was used to immunoprecipitate skin tissue extracts. These results are representative of five independent experiments. Journal of Investigative Dermatology 2001 117, 971-976DOI: (10.1046/j.0022-202x.2001.01473.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Time course of insulin-stimulated phosphorylation of MAPK and Akt in the skin of intact normal fasted rats. Whole tissue extracts from rats injected with saline (–, 0 min) or insulin (+, 1, 3, and 5 min) were prepared as described in Materials and Methods. Nitrocellulose membranes were immunoblotted with αphospho-MAPK (1 µg per ml) (A) or αphospho-Akt (B). For controls (last lane in each blot), IgG from normal rabbit serum was used to immuno precipitate skin tissue extracts. These results are representative of five independent experiments. Journal of Investigative Dermatology 2001 117, 971-976DOI: (10.1046/j.0022-202x.2001.01473.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions