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Fig. 8. Analysis of geminin splice variants in human cell lines and tissues and detection of Geminin and Cdc6 proteins in testis but not ovary. (A) Detection of two geminin isoforms after RT-PCR of total RNA in cell lines using geminin-specific primers. In the first lane a control PCR without DNA is shown. Actin was amplified as a positive control for each sample. (B) Detection of geminin isoforms after RT-PCR of total RNA in human tissues as described above. Actin was amplified as a positive control for each sample and a control PCR without actin was negative as above (data not shown). (C) Amplification plot resulting from quantitative real-time RT-PCR of Geminin mRNA splice variant GemE and endogenous control 18s rRNA (inset) in RNA from human testis, ovary, colon and cervix. Dashed lines represent RNA from human testis whereas solid lines represent RNA from human ovary, colon and cervix. (D) Immunoblots of Geminin in human testis and ovary tissue lysates (75 µg each/lane) with affinity-purified antibodies G94 and G95 alone, or after preincubation with recombinant Geminin. A band of approximately 20 kDa (arrow) was also detected with both G94 and G95. This interaction was blocked after preincubation of the antibodies G94 and G95 with recombinant Geminin. (E) Immunoblots of total cell lysates from testis and ovary with an anti-Cdc6 antibody.
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