Figure 3

The short telomere phenotype of the yku70-R456E mutant cannot be rescued by overexpressing TLC1. (A) TLC1 RNA overexpression (OE) was confirmed by northern blotting of WT, yku70-R456E, and yku70Δ strains transformed with a 2 micron TLC1 plasmid (TLC1 OE) or a 2 micron empty vector (EV) following 1X and 4X streak outs. Blots were also probed with ACT1 for loading. Full-length blots are presented in Supplementary Fig. S8. (B) Quantification of northern blot shown in A. TLC1 levels were quantified relative to ACT1 RNA and normalized to WT EV 1X streakout cells. (C) Telomere length analysis by Southern blotting of 1X-4X serial streakouts of WT, yku70-R456E, and yku70Δ strains harboring 2 micron TLC1 OE or EV plasmids. The full-length blot is presented in Supplementary Fig. S9. (D) Northern blot probed for TLC1 RNA in cytoplasmic and nuclear fractions (top) and western blot analyzed for tubulin, which is a cytoplasmic protein. The absence of tubulin the nuclear fraction indicates the nuclear prep was free of cytoplasmic contamination. The full-length northern blot and western blot are presented in Supplementary Fig. S10. *Presumed nonspecific band (see retention of a similarly migrating band in the tlc1∆ lane of full-length northern blot of Fig. 2B which is presented in Supplementary Fig. S7).