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determine whether this transient nature of Snail mRNA expression is reflected at the protein level, we generated an antibody against the N-terminal sequence that resides upstream of the more conserved zinc finger domains. As judged by Western blot analysis, the antibody did not detect endogenous proteins from cultured keratinocytes, but it did yield a band of the expected size from keratinocytes transiently expressing a hemagglutinin (HA)-tagged Snail protein (Figure 1B). The antibody also recognized a band corresponding to the size of endogenous Snail (approximately 28 kDa) in lysates from embryonic mouse skin, the temporal appearance of which corresponded to the waves of hair follicle morphogenesis from E15.5 to newborn when over 90% of the hair on the mouse is formed (Figure 1B). Consistent with the Western blot data, immunohistochemical analysis did not detect Snail in single-layered E13.5 epidermis (Figure 1C) nor in the placode, which is the earliest morphological sign of the commitment of multipotent cells of the embryonic ectoderm to a hair cell fate (Figure 1D). Consistent with the in situ hybridization results, anti-Snail antibody labeled only hair buds and not follicles at more mature stages of development (Figure 1E). Taken together, the anti-Snail antibody appeared to be specific for its target protein. It did not detect other Snail family members known to be expressed in keratinocytes and/or skin (unpublished data). Furthermore, the immunohistochemical data paralleled our Snail in situ hybridization data revealing transient Snail expression at the hair bud stage (Figure 1A)."}