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Fig. 3. Analysis of Wt1 protein in Y-79 cells. Twenty µg of protein in total lysates obtained from undifferentiated Y-79 cells and from cultures containing ~60% neuron-like cells were separated on a 10% SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-Wt1 antibody (A). Down-regulation of Wt1 protein in differentiated Y-79 cells was confirmed by immunocytochemistry. A representative example of Wt1 immunostaining is shown in C. Note the strong labeling of the round-shaped undifferentiated Y-79 cells, whereas the neuron-like cell (arrowhead) does not stain with anti-Wt1 antibody (C). B shows a phase contrast view of C to make the process-bearing (Wt1-negative) cell more visible (arrow). No specific staining was obtained when the primary anti-Wt1 antibody was replaced by normal rabbit serum (phase contrast view in D and E).