FIGURE 2.

Proteolytic processing of FAT1 cadherin in keratinocytes. Cell lysates of HaCaT keratinocytes were analyzed for FAT1 expression by Western blotting using the FAT1 C-terminal pAbs after seeding equal numbers of cells into different sized culture dishes and growing for 2 days (A) or seeding equal numbers of cells into the same size culture dish and growing for 1–4 days (B). All cultures were lysed in an equal volume, and an equal proportion of the lysate was applied to the gels. Membranes were then reprobed with anti-GAPDH as a loading control. The high molecular mass band observed at the expected ∼500-kDa size of FAT1 is indicated by an arrow together with a prominent band at ∼85 kDa. C, HaCaT keratinocytes were pulse-labeled with [³⁵S]cysteine/methionine and subjected to chase in unlabeled medium for 0–360 min. At each indicated time point, cells were collected, lysed, and immunoprecipitated using a mixture of anti-FAT1 mAbs (NTD-7 and CTD-7). Panel i, the samples were then resolved on 3–8% Tris acetate gels that were subsequently dried and subjected to storage phosphorimaging. The asterisk indicates a nonspecific (ns) band also observed in control immunoprecipitates throughout the chase (not shown). Panel ii, densitometric analysis demonstrating the relative expression of p500 to the p430 and p85 bands that reflects the proteolytic processing events occurring in FAT1 (see text for further details).