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Fig. 2. A, Wt1 protein in different cell lines and in kidneys from 3-day-old rats (P3). Twenty µg of protein from total cell lysates were loaded per lane and separated on a 10% SDS-PAGE. Immunoblotting was performed with polyclonal rabbit anti-Wt1 antibody. The membrane was reprobed with a polyclonal anti-ß-actin antibody to detect differences in protein loading. Note the high Wt1 protein content (Mr 55,000 band) in retinoblastoma-derived Y-79 cells as compared with HEK293 cells and 3-day-old rat kidney. Wt1 protein levels were similar in Y-79 and in K562 leukemia cells. B, analysis of mRNA levels in undifferentiated Y-79 cells and in cultures containing >60% neuron-like cells. Wt1 transcripts were dramatically down-regulated in differentiated versus undifferentiated cells as demonstrated by semiquantitative RT-PCR (B). In contrast, mRNA levels of ß-actin and the AP-2 transcription factor were similar in undifferentiated and neuron-like Y-79 cells (C). undiff., undifferentiated; diff., differentiated.