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. 2006 Mar 27;172(7):1093–1105. doi: 10.1083/jcb.200507111

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Copyright © 2006, The Rockefeller University Press

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Figure 4. — In vitro and in vivo human skin profiles of TβRs (I, II, and III) and their transmission of TGFβ-stimulated Smad_2/3 activation. (A) Equal amounts of cell lysates of serum-starved HKs, MCs, DFs, and HDMECs were resolved in three SDS gels and subjected to Western blot analyses with antibodies against TβRI/ALK5 (a), TβRII (c), or TβRIII/betaglycan (e). Each lower part of the three membranes was blotted with an anti–β-actin antibody as a control (b, d, and f). The expression was quantitated by a densitometry scan, giving rise to a ratio of TβR band intensity over its corresponding β-actin band intensity. (B) Normal human skin sections were subjected to indirect immunofluorescence staining with the antibodies against TβRI (a), TβRII (f), TβRIII (k), DAPI (b, g, and l), rhodamine-conjugated phalloidin (c, h, and m), or corresponding IgG controls (see Materials and methods). The images show skin tissue distribution of the TβRs (a, f, and k), nuclei (b, g, and l), F-actin (c, h, and m), and all three stains merged (d, i, and n). A section of the anti-TβRI antibody staining (a, dotted box) was enlarged in the inset to visualize cellular levels of staining. Bar, 20 μm. (C) The cells were either untreated or treated with the indicated concentrations of TGFβ3 for 15 min, and equalized cell lysates (40 μg/lane) were subjected to Western blot analyses with either antiphospho-Smad_2/3 (a, c, and e) or anti-Smad_2/3 (b, d, and f) antibodies. (D) The cells were either untreated or treated with 2.5 ng/ml TGFβ3 for the indicated periods of time. Equalized cell lysates were subjected to Western blot analyses with either antiphospho-Smad_2/3 (a, c, and e) or anti-Smad_2/3 (b, d, and f) antibodies.