Nature research paper: CDC7-independent G1/S transition revealed by targeted protein degradation
, Quantification of Cdk1 phosphorylation on threonine-161 and threonine-15 , using mass spectrometry. Cdc7/Tir1 ESC were treated with auxin and analyzed after 5, 24 and 48 h. Black dots, phosphorylation levels at time 0./Tir1 ESC treated with auxin or vehicle for 24 h, and used for kinase reactions with [γ, Recombinant Mcm2 was phosphorylatedwith recombinant CDK1-cyclin B1 or CDC7-DBF4 and incorporation of phosphate into serine-27 quantified by mass spectrometry.
At the indicated time-points after stimulation, cells were stained with propidium iodide and analyzed by flow cytometry. SS, serum starved cells./Tir1 ESC were synchronized in mitosis by nocodazole and released . Upon release, Cdc7 degradation was induced by auxin addition to account for degradation time. Two h later, when cells reached G1 phase , they were treated with Cdk1 inhibitor .
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