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. 2010 Jan 29;37(2):159–171. doi: 10.1016/j.molcel.2009.12.023

Figure 2.

Figure 2

HtrA2 Cleaves WT1 in Cells

(A) HeLa cells were cotransfected with plasmids encoding WT1 (1 μg) and vector control, along with plasmid driving expression of either wild-type or the mutant HtrA2 derivative S306A (0.5 μg). At 24 hr after transfection, whole-cell lysates were prepared and immunoblotted with the indicated antibodies. The full-length and processed forms of HtrA2 are indicated. Nonspecific bands are indicated by asterisks.

(B) HeLa cells were cotransfected with plasmids encoding GFP-WT1-KTS, GFP-WT1 + KTS, or GFP (1 μg) in combination with vector control, wild-type, or mutant HtrA2 (0.5 μg) and 24 hr after transfection were analyzed as in (A). Below, a diagram of WT1 is shown, indicating the transcriptional regulatory and zinc finger regions. The epitopes recognized by the WT1 N-Ter and C-Ter antibodies and also the approximate locations of HtrA2 cleavage sites are indicated.

(C) HeLa cells were transfected as in (B), and then nuclear (Nu) and cytoplasmic (Cyt) extracts were prepared. The samples were immunoblotted with the antibodies indicated. The GFP-WT1 N-terminal and C-terminal proteolytic fragments are indicated.