For temperature-shift experiments, following CuSO4 addition, cells were shifted to the indicated temperatures for the remainder of the assay.
(A ) Northern blot analysis of inv1 expression in the indicated strains after an hour shift from high glucose (2%) to low glucose (0.1%).
(F ) Northern blot analyses of inv1 expression in the indicated strains after an hour shift from high glucose (2%) to low glucose (0.1%).
Cells were then shifted to the indicated temperature for 30 min. To release from G1 arrest, the cells were washed 3 times with warm water and then resuspended in pre-warmed medium at the indicated temperature.
Overnight cultures were diluted and allowed to grow for several hours to reenter log phase and subsequently shifted to the indicated temperatures for 3 hr.
Wild-type (A22678) and ndc80-1 (A28221) cells were induced to sporulate at 25°C. 2 hr 30 min after transfer into sporulation medium, cells were shifted to the indicated temperature and sporulation efficiency was determined after 24 hr.
See also Figure 4 figure supplement 3. DOI: http://dx.doi.org/10.7554/eLife.04120.010 10.7554/eLiFigure20.011 Figure 4 figure supplement 1. Gel-shift assays with the indicated Rev mutants.
Formation of the SDS-resistant PA63 7) pore species was detected by immunoblotting as previously described [24], except cells were incubated with 2.5×10−8M PA and 2×10−9M LF for 2 hours at 4°C, washed and shifted to 37°C for the indicated times.
Worm extracts were made from the indicated daf-2 and wild-type strains by shifting 200 L4s from 15 to 25 °C and allowing them to grow for 2 days.
