Fig. 2. Analysis of TBP family triple knockdown (ablation of tbp, tlf and tbp2 mRNA). (A) Morphology of TKD embryos at stage 10.5, relative to water-injected control (left panel). (B) Control experiments verified efficient TBP protein depletion (western blot, left panel) and knockdown of tlf and tbp2 transcripts by RT-qPCR in TKD embryos relative to control (Ctrl). Tubulin (western) and gapdh serve as controls. (C) Expression levels of TFI gene transcripts (left, in blue) and transcripts that require TBP, TBP2 or TLF (light gray) in TKD embryos relative to control embryos, as determined by RT-qPCR. gapdh transcript levels (dark gray) served as control. (D) Box plots showing fold change (log2) of transcript levels of all expressed genes, decreased genes (DEseq FDR 0.1, see Materials and Methods) and TFI genes, as determined by RNA sequencing of ribosomal RNA-depleted samples of TKD embryos versus control embryos. (E) Recruitment of initiating RNAPII-Ser5 determined by ChIP-qPCR. 5′ and 3′ ends of TFI genes were analyzed. Comparison of signals obtained from control (light blue) and TKD embryos (dark blue) indicates significant recruitment of RNAPII-Ser5 to TFI genes under TKD conditions.
Image published in: Gazdag E et al. (2016)
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