Coordination between discrete Mitotic Arrest Deficient 1 (MAD1) domains is required for efficient mitotic checkpoint signaling

Wenbin Ji; Yibo Luo; Ejaz Ahmad; Song-Tao Liu

100mM KCl, 1mM DTT, 30% glycerol. Concentrations of recombinant proteins were determined by comparing the target band with BSA standards on Coomassie blue stained gels using ImageJ software (66) . In vitro binding assays. 4µl of 5× binding buffer (100mM Tris-HCl, pH 8.0, 750mM NaCl, 50mM MgCl 2 , 2.5% NP-40, 50% glycerol, 500 µg/ml BSA) was mixed with recombinant GSTtagged MAD1 fragments and MAD2 mutants or The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/212704 doi: bioRxiv preprint pelleted and washed 4 times with wash buffer (1× PBS, pH 7.4, 150 mM NaCl, 0.5% NP-40, 10% glycerol). Then 10µl 2× SDS sample buffer was added to the beads and the samples were heated at 80C for 10 min before SDS-PAGE. The gel was transferred to PVDF membranes (Millipore) for immunoblotting. In vitro kinase assays. GST-tagged MPS1 kinase was purchased from Invitrogen or was purified from Sf9 cell lysates as well as His-tagged MPS1 (64) . Myelin basic protein (MBP) was purchased from Sigma as an artificial substrate for MPS1. In vitro kinase assays were set up similarly as previously described (28, 64) : 4 µl of 5× kinase buffer (125 mM Tris-HCl, pH 7.5, 300 mM βglycerophosphate, 50 mM MgCl 2 ) was mixed with recombinant kinase, substrates, 5µCi 32 P -ATP and 50 µM cold ATP. In some reactions, MPS1 kinase inhibitors reversine (Calbiochem) or AZ3146 (Selleckchem) was used at 500 nM and 2 µM final concentrations, respectively. DMSO concentration is kept below 0.5%. H 2 O was added to make the final volume of 20 µl. The reactions were incubated at 30C for 30 min and then terminated by adding 20 µl 2×SDS sample buffer. Samples were subjected to SDS-PAGE followed by Coomassie staining. After destaining, the SDS-PAGE gel was vacuum dried. Phosphorylation of the substrates was visualized by autoradiography. Samples for mass spectrometry were prepared using only 0.5 mM cold ATP in the kinase reactions and separated on SDS-PAGE. Phosphorylated residues on the excised bands were determined by mass spectrometry (MSBioworks, Ann Arbor).