Gene Recruitment of the Activated INO1 Locus to the Nuclear Membrane (original) (raw)
Figure 2
Ino2/Ino4 Bind to the INO1 Promoter Constitutively
(A) Untagged control cells (upper images), or cells in which the endogenous copies of INO2 and INO4 were replaced with HA-tagged Ino2 (center images) or HA-tagged Ino4 (lower images) were harvested in mid-logarithmic phase and washed into medium with or without _myo_-inositol. After 4.5 h, about 1.5 × 108 cells were harvested and processed for Northern blot analysis (light images with dark bands, right). Northern blots were probed against both INO1 and ACT1 (loading control) mRNA. The remaining cells were fixed with formaldehyde and lysed. Chromatin was sheared by sonication and then subjected to immunoprecipitation with anti-HA agarose. Input DNA (In) and immunoprecipitated DNA (IP) were analyzed by PCR using primers to amplify the INO1 promoter and the URA3 gene. Amplified DNA was size-fractionated by electrophoresis on ethidium bromide-stained agarose gels (dark images with light bands, left).
(B) Quantitative PCR analysis. Input and IP fractions were analyzed by real-time quantitative PCR. The ratio of INO1 promoter to URA3 template in the reaction is shown. Error bars represent the standard error of the mean (SEM) between experiments.