Figure 4

Reversibility of temperature-induced dADAR and editing levels. (A) Western blot analysis of the HA-tagged dADAR after three days at 10°C, 20°C and 30°C and after being shifted from 30°C to 20°C for 24, 48, or 72 hours. β-actin is presented as a loading control. Wild type dADAR, which lacks the HA tag is presented as a negative control (−HA). (B) Quantification of western blot analysis. dADAR-HA signal is normalized to the β-actin signal from each lane. Bars represent standard deviation. (C) Editing at specific sites after temperature shift. Editing after 72 hours at 30°C is depicted in red. After animals were held at 30°C for 72 hours they were shifted to 20°C for 24 (white), 48 (gray) and 72 (dark gray) hours. Animals held at 20°C for 72 hours but not shifted are depicted in black. Editing of synaptotagmin-1 sites 2 to 4 is unresponsive to temperature (Figure 1B) yet editing at sites 2 and 3 significantly decreases after 24 hours at 20°C. These sites recover to near-20°C levels after 48 and 72 hours. Editing of paralytic sites 1 to 3 begins to recover from 30°C levels after just 24 hours. Editing at shab site 6 increases at elevated temperature (Figure 1C) and after 72 hours recovers to near-20°C levels. Shab site 7, edited to 100%, is unresponsive to temperature (Figure 1D) and recovery. Bars represent standard error. P <0.0001: **, P < .05: *, not significantly different: NS.