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. 2005 Aug;25(16):6879–6888. doi: 10.1128/MCB.25.16.6879-6888.2005

FIG. 4.

FIG. 4.

Effect of expression of SAVEE-SLBP on regulation of histone mRNA. Five micrograms of total RNA from HeLa cells stably expressing His-tagged WT SLBP (A) or His-tagged SAVEE-SLBP (B) was analyzed by an S1 assay using a mixture of the human replication-dependent H2a gene and the H3.3 gene as a probe. The cells were untreated cells (lane 1) or cells that were treated with HU for 30 min (lane 2), 1 h (lane 3), 2 h (lane 4), 3 h (lane 5), or 4 h (lane 6) or with CH for 1 h (lane 7). Note that the relative intensities of the H3.3 and H2a protected fragments vary between different experiments because of different relative specific activities of the probes. (C) Lysates from HeLa cells expressing SAVEE-SLBP that were not transfected with any siRNA (lane 1) or transfected with an siRNA that specifically targets the endogenous WT SLBP once (lane 2) or twice (lane 3) were prepared 48 h after the final transfection. Ten micrograms of total protein was resolved by 10% SDS-PAGE and analyzed by Western blot assay with anti-SLBP antibody. (D) Five micrograms of total RNA from HeLa cells stably expressing His-tagged SAVEE-SLBP that were not transfected with any siRNA (lanes 1 to 3) or were transfected with SLBP siRNA either once (lanes 4 to 6) or twice (lanes 7 to 9) was analyzed by S1 nuclease assay using a mixture of histone H2a and H3.3 genes as a probe. The cells were either untreated (lanes 1, 4, and 7) or treated either with HU for 45 min (lanes 2, 5, and 8) or with CH for 45 min (lanes 3, 6, and 9) 48 h following the final transfection.

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