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Figure 5 | Genome Biology

Figure 5

From: The transcriptional network activated by Cln3 cyclin at the G1-to-S transition of the yeast cell cycle

Figure 5

Experimental validation of predicted targets. ChIP assays with Mbp1TAP and Swi4TAP were carried out for a number of targets for which TF binding had not been detected before. (a) PCR products for predicted targets ELG1, STB1, VRG4 and STU2 are shown. Cells were grown either asynchronously or enriched in G1 with α factor. Three dilutions (1:1,500, 1:4,500, 1:13,500 for tagged strains; 1:2,500, 1:7,500, 1:22,500 for untagged strains) of the whole cell extract (WCE) and two (1:5, 1:15) of the immunoprecipitates (IP) were used. PCR was carried out for 28 or 30 cycles for tagged and untagged strains, respectively. As an internal control for non-specificity the gene DYN1 was used. The PCR product amplified from this gene was several kilobases away from the closest promoter. (b) Quantification of ChIP assays. Optical density of bands was measured with ImageJ. The relative enrichments shown are calculated as ratios of specific to non-specific (DYN1) products in the IP compared to the input (WCE). Two independent PCRs were carried out per gene tested (just one PCR in the untagged strains). The average and standard deviations (error bars) of two or three different exposures are shown. Genes ELG1, SLD2, STB1 and CDC45 (positive control) were tested in the Mbp1TAP ChIP; genes STU2, ERP2, VRG4 and SVS1 (positive control) were tested in the Swi4TAP ChIP.

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