Magicplot Pro Product Keyl ((HOT))

Magicplot Pro Product Keyl ((HOT))

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A) Immunoblot analysis of UbTFΔme/wt and its various point mutation variants with antibody directed against the N-terminal domain of UbTF. A schematic diagram of the different proteins is given in the text, indicating the relative transformation of the UbTCP, UbTTP and UbTLP phenotypes of these variants by site-directed mutagenesis. B) A fraction of mutant UbTF variants were shown to be present as heterotetramers (lanes 1 and 2) or single copies (lanes 3 and 4) in co-transfection experiments. The protein lysates were precipitated with anti-Flag antibodies and immunoblotted with antibody against UbTF used in the original western blot in order to verify similar expression levels of the various UbTF variants. A non-specific band is also shown to indicate equal total protein loading in the experimental lane.](pone.0076407.g006){#pone-0076407-g006} Magicplot Pro Product Keyl A) Protein sequence of UbTF showing coding exon 6, the differentially spliced coding exon 7 and coding exon 8 in black and the intervening introns in red. The E210K mutation and the positions of the potential splice branch sites of the relevant introns in the E210K alleles are indicated in grey. B) Diagram illustrating the luciferase oligo species that were used in the splicing assays performed in the presence of different UbTF variants. The plasmid used for expression of the different WT, mutant or RNase H mutant UbtfsLuc resulted in the synthesis only of single luciferase species (LUC) due to multiple rounds of the different reaction phases to form pre-mRNA (PRE) and mature mRNA (MAT) containing exons 1 to 7 of the LUC gene. The full length luciferase protein (FL) amounts contained the first seven exons of the LUC gene as well as the first and second introns of LUC together with the remaining exons 1 to 7 of the LUC gene. In this experiment, exon 1 was removed from the LUC gene by complementarity to the oligo used to transcribe RNA containing exons 1 to 7.

A) RNA metabolic pulse labelling to reveal 47S pre-rRNA synthesis and processing products in UbtfE210k/E210K knock-in and wild type Ubtfwt/wt MEFs. Gel fractionation of RNA after increasing labelling times is shown for two individual (numbered) MEF isolates. B) DNA base sequence of the differentially spliced region of mouse Ubtf gene showing coding exon 6, the differentially spliced coding exon 7 and coding exon 8 in black and the intervening introns in red (taken from GRCm38:11:102303960:102320342). The position of the G>A gene mutation, the cause of the E210K change in the UBTF protein, is indicated as are the potential splice branch sites in the intervening introns that most closely fit the yTnAy consensus [53]. d2c66b5586