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Fig. 6. Overexpression of treacle protein and rDNA transcription. (A) HeLa cells were transfected with constructs that expressed GFP alone or GFP fused with full-length (amino acids 1-1488) and deletion mutant forms of treacle. (Right) The cellular localizations of GFP and treacle-GFP fusion proteins are shown. (B) HeLa cells transfected for 24 h with the constructs shown in A were metabolically labeled with ³²P as described in Materials and Methods. Total RNA was isolated from cells labeled for 1.5 h, resolved on 1% agarose-formaldehyde gel (~1 mg per lane), blotted onto Zeta-probe membrane, and analyzed by phosphorous imaging. The membrane was stained with 0.2% methylene blue to check RNA loading. Comparison of the ³²P image and methylene blue staining shows that the differences in the ³²P signals are mostly caused by differences in the amount of labeled RNA.