Fig. 3

Optimization of split CRY2/CIB1 transcriptional system. (A) Schematic and immunostaining of new CRY2-BD construct (CRY-Gal(1?65)) assayed in split transcriptional system. HEK293T cells expressing CRY2-Gal(1?65) were kept in dark or exposed to blue light pulses for 18 h, then assayed for immunohistochemistry using an anti-Gal4BD antibody. (B) Luciferase activity of HEK293T cells expressing indicated constructs and a GalUAS-luciferase reporter and exposed to dark or blue light pulses as in (A) for 18 h. Data represents average and error (s.d., n = 3). Experiments were repeated two times with similar results. (C) Comparison of activation domain fusions. HEK293T cells were transiently transfected with CRY2-Gal(1?65) and indicated AD-fusion constructs, along with a GalUAS-luciferase reporter and tested for activity as in (B). Data represents average and error (s.d., n = 3). Experiments were repeated three times with similar results. (D) Immunoblot of HEK293T cells expressing a GalUAS-GFP-HA reporter with CRY2-Gal(1?65) and CIB1-VP64 incubated in dark or blue light for 18 h. (E) Spatial regulation. HEK293T cells expressing CRY-Gal(1?65), VP64-CIB1, and a GalUAS-GFP reporter were exposed to 18 h patterned blue light, followed by imaging of GFP reporter expression. Scale bar, 1 cm. (F) Dose-dependent regulation. HEK293T cells expressing constructs as in (E) were illuminated with varying light intensities for 18 h, followed by immunoblotting using an anti-GFP antibody or anti-tubulin control.