Figure EV1. SNF1 complex is required for timely checkpoint activation.

1. DNA content was monitored by flow cytometry. Cells were grown in media containing 2 or 5% glucose and exponential cells were arrested into G1 phase by alpha‐factor before release into corresponding fresh media for the indicated time.
2. Rad53 hyperphosphorylation of WT and snf1Δ in response to HU. Cells were synchronized in G1 and released into the fresh medium (5% glucose) containing 200 mM HU for the indicated time. Cell extracts were prepared for immunoblotting against anti‐Rad53 antibodies. Tubulin was probed as a loading control.
3. 5% glucose cannot rescue the HU sensitivity of snf1Δ. WT and snf1Δ strains were grown and spotted on HU plates with 5% glucose. All plates were cultured at 30°C for 48 h before photography.
4. H2A S129 phosphorylation (namely γ‐H2A) of WT and snf4Δ in response to HU. γ‐H2A was detected by immunoblotting (upper panel). Three independent experiments were performed and quantified by Image J (lower panel). Error bars represent standard deviations (SD) from three biological repeats.
5. Rad53 phosphorylation of WT and snf4Δ in response to HU. Rad53 phosphorylation was detected as above. Mcm2 was probed as a loading control.

Source data are available online for this figure.