Figure 1. TEV-mediated cleavage of Barren disrupts condensin I function within a few minutes.
(a) Schematic representation of condensin complex indicating the position of the 3xTEV cleavage sites in the kleisin subunit Barren (aa175). (b) In vitro cleavage of BarrenTEV-myc. Extracts were prepared from ovaries of flies expressing solely TEV-cleavable Barren and incubated with TEV protease for the indicated time points (periods of time). The presence of full-length and cleaved Barren was monitored by western blot using myc antibodies. Tubulin was used as loading control. (c) Early embryos (0–30 min old) expressing HisH2AvD-mRFP1 (red) were injected with mRNA coding for BarrenTEV-EGFP (green). Embryos were aged for 1 hr-1hr 30m to allow for protein expression. Embryos were injected with 12 mg/ml UbcH10C114S protein to arrest in metaphase and subsequently with TEV-protease; images depict the same region before and after TEV injection; times (minutes:seconds) are relative to the time of injection; scale bar is 10 μm.
Figure 1—figure supplement 1. A TEV-cleavable system to destroy condensin I.
