Fig. 2. SPRR4 is a CE precursor protein. (A) Subcellular location of SPRR4-GFP and GFP in living cells. Monolayers of normal human keratinocytes were transfected with either SPRR4-GFP (1,2) or GFP (3,4), as a control. Cultures were either maintained in medium without calcium (1,3) or induced to differentiate for 40 hours in 1.8 mM Ca²⁺ (2,4). Cells were analyzed with a laser scanning confocal microscope. (B) Isolation of cornified envelopes from keratinocytes transfected with SPRR4-GFP. Transfected cells were incubated for 48 hours with A23187 ionophore, treated with 2% SDS and 10 mM DTT and examined using a fluorescence microscope (1, fluorescence; 2, phase-contrast). The middle image in panels 1 and 2 shows a rigid envelope and the flanking images show fragile envelopes. Bar, 15 µm.