Prevalence of K5 and K14 disulfide bonding in wild-type newborn mouse skin keratinocytes in primary culture. (A) Immunoblotting analysis shows the occurrence of multiple disulfide-bonded species immunoreactive for K5 and K14 epitopes. M, keratin monomer; D, keratin dimer species. The asterisk denotes cross-reactivity of anti-K14 antibody with K5. (B) Analysis of the solubility of disulfide-bonded K5- and K14-containing species using detergent extraction and Western immunoblotting analysis. Sol, Triton-soluble fraction; Ins, Triton-insoluble fraction. (C) Immunofluorescence-based imaging for keratin (green channel) and disulfides (red channel) in mouse keratinocytes under low and high calcium culture conditions. The middle and bottom rows show magnified views of the boxed areas in the top row. The bottom row shows the spatial overlap between K5/K14-specific signals and the global signal for disulfide bonds. S-S, disulfide bonds. Arrows depict colocalization between K5/K14 and S-S in the perinuclear region. Arrowheads indicate colocalization at the cell–cell interface. Bar, 10 µm.