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Fig. 1 | BMC Biotechnology

Fig. 1

From: Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway

Fig. 1

Screening of a Medaka cDNA library. a Schematic drawing of the screening process. 24 clones of the cDNA library were pooled and all pools were arrayed in 96-well plates. P53-negative H1299 cells were transfected in 96 well-plates with p53 (5 ng), mdm2 (45 ng), one of the cDNA library pools (150 ng) and myc-ror2 (5 ng) as control. To monitor Mdm2 activity, and Mdm2 and p53 abundance in the absence of co-transfected pools of the cDNA library, 2 out of 14 wells were transfected with p53 and myc-ror2 (control 2) or with p53 and mdm2 and myc-ror2 (control 1) without library pools. For transfection, total amounts of plasmid DNA in all samples were adjusted to 205 ng using vector DNA. 24 h after transfection, cells were lysed and abundance of p53, Mdm2 and Myc-ROR2 were determined by Western Blotting. Abundance of PCNA was monitored for loading control. b H1299 cells were transfected with plasmids encoding p53, Mdm2 and Myc-ROR2 together with the indicated pools of the cDNA library, without the library (ctrl 1) or without the library and without the plasmid encoding Mdm2 (ctrl 2), for control. 24 h after transfection, cells were lysed and abundance of p53, Mdm2, Myc-ROR2 and PCNA were determined by Western Blotting

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