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Figure 1 | Journal of Molecular Signaling

Figure 1

From: E4BP4 facilitates glucocorticoid-evoked apoptosis of human leukemic CEM cells via upregulation of Bim

Figure 1

Primer Specificity for human and mouse E4BP4 and generation of M.E4BP4 expressing CEM C1-15mE#3 cells: Panel A: Sequence alignment of partial mouse and human E4BP4 coding sequences within exon 2 (numbering is based on +1 for the first nucleotide of the coding region). Upper rows indicate mouse E4BP4 sequence in upper case and lower rows show human E4BP4 sequence in lower case. Forward and reverse primers are underlined with solid and dotted lines respectively (mE4BP4 = 742/1117 and hE4BP4 = 742/991). Panel B: The top gel shows specificity of mE4BP4 primers for M. E4BP4 template. PCR using DNA extracted from human CEM clones failed to amplify any product. The positive control template of plasmid pCR3.1-mE4 amplified a 376bp fragment. For the lower gel, 1:100 and 1:1000 dilutions of the plasmid DNA pCR 3.1-mE4BP4 were subjected to qRT-PCR analysis using primers mE4BP4 (742/1117) and hE4BP4 (742/991). Product amplification was observed only with mE4BP4 (742/1117). Panel C: CEM-C1-15 cells electroporated with 13.5 μg of linearized pCR3.1-mE4 plasmid expressing M. E4BP4 were selected in the presence of 400 μg/ml Geneticin. Seven micrograms of total RNA extracted from two mass culture populations (mE*a and mE*b) and four clones (mE#1-mE#4) obtained by limiting dilution was subjected to reverse transcription followed by end-point PCR analysis for the expression of M. E4BP4 (top gel). The clone labeled mE#3 (CEM C1-15 mE#3) (circled) was used for further analysis. Lower gel shows PCR products obtained with both mE4BP4 (742/1117) and hE4BP4 (742/991) primers using reverse transcription products from clone mE#3, parental CEM-C1-15 and CEM C7-14 cells as the template.

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